Chimeric, human and humanized anti-CSAP monoclonal antibodies

ABSTRACT

The present invention provides humanized, chimeric and human anti-CSAp antibodies and anti-CSAp antibody fusion proteins that are useful for the treatment and diagnosis of various cancers, including colon cancer.

This application claims priority to PCT/US02/10235 filed Apr. 3, 2002.This application is also a continuation of U.S. Ser. No. 10/116,116,filed Apr. 5, 2002, now U.S. Pat. No. 7,387,772, which is acontinuation-in-part of U.S. Ser. No. 09/823,746, filed Apr. 3, 2001,now U.S. Pat. No. 6,962,702, which is a continuation-in-part of U.S.Ser. No. 09/337,756, filed Jun. 22, 1999, now U.S. Pat. No. 7,074,405,which claims priority to U.S. Ser. No. 60/104,156, filed Oct. 14, 1998,now expired, and U.S. Ser. No. 60/090,142, filed Jun. 22, 1998, nowexpired, the contents of all of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to immunological reagents for therapeutic use, forexample, in radioimmunotherapy (RAIT) and chemoimmunotherapy, anddetection and/or diagnostic uses, for example, in radioimmunodetection(RAID), ultrasonography, and magnetic resonance imaging (MRI). Inparticular, the invention relates to naked antibodies (unconjugated) anddirectly-conjugated antibodies, as well as bi-specific antibodies(bsAbs) and bi-specific antibody fragments (bsFabs) which have at leastone arm which is reactive against a targeted tissue and at least oneother arm which is reactive against a linker moiety. Further, theinvention relates to monoclonal antibodies that have been raised againstspecific immunogens, being human, humanized and chimeric monoclonalantibodies, as well as human, humanized and chimeric bi-specificantibodies and antibody fragments having at least one arm which isreactive against a targeted tissue and at least one other arm which isreactive against a linker moiety, DNAs that encode such antibodies andantibody fragments, and vectors for expressing the DNAs.

The present invention also relates to humanized, chimeric and humananti-CSAp antibodies, particularly monoclonal antibodies (mAbs),therapeutic and detection/diagnostic conjugates of humanized, chimericand human anti-CSAp antibodies and methods of diagnosing/detecting ortreating a malignancy using humanized, chimeric and human anti-CSApantibodies. The present invention also relates to antibody fusionproteins or fragments thereof comprising at least two anti-CSAp mAbs orfragments thereof or at least one anti-CSAp mAb or fragment thereof andat least one second mAb or fragment thereof, other than the anti-CSApmAb or fragment thereof. The humanized, chimeric and human anti-CSApmAbs, fragments thereof, antibody fusion proteins thereof, or fragmentsthereof may be administered alone, as a therapeutic conjugate or incombination with a therapeutic immunoconjugate, with other nakedantibodies, or with other therapeutic agents or as adiagnostic/detection conjugate. The present invention also provides DNAsequences encoding humanized, chimeric and human anti-CSAp antibodies,and antibody fusion proteins, vectors and host cells containing the DNAsequences, and methods of making the humanized, chimeric and humananti-CSAp antibodies.

2. Related Art

An approach to cancer therapy and detection/diagnosis involves directingantibodies or antibody fragments to disease tissues, wherein theantibody or antibody fragment can target a detection/diagnostic agent ortherapeutic agent to the disease site. One approach to this methodologythat has been under investigation involves the use of bi-specificmonoclonal antibodies (bsAbs) having at least one arm that is reactiveagainst a targeted diseased tissue and at least one other arm that isreactive against a low molecular weight hapten. In this methodology, absAb is administered and allowed to localize to target, and to clearnormal tissue. Some time later, a radiolabeled low molecular weighthapten is given, which being recognized by the second specificity of thebsAb, also localizes to the original target. The same technology can beused to target therapeutic isotopes, drugs and toxins selectively todiseased tissues, particularly cancers against which the bsAb istargeted, or non-radioactive diagnostic agents for improved diagnosisand detection of pathological lesions expressing the target antigen.

Although low MW haptens used in combination with bsAbs possess a largenumber of specific imaging and therapy uses, it is impractical toprepare individual bsAbs for each possible application. Further, theapplication of a bsAb/low MW hapten system has to contend with severalother issues. First, the arm of the bsAb that binds to the low MW haptenmust bind with high affinity, since a low MW hapten is designed to clearthe living system rapidly, when not bound by bsAb. Second, thenon-bsAb-bound low MW hapten actually needs to clear the living systemrapidly to avoid non-target tissue uptake and retention. Third, thedetection and/or therapy agent must remain associated with the low MWhapten throughout its application within the bsAb protocol employed.

Of interest with this approach are bsAbs that direct chelators and metalchelate complexes to cancers using Abs of appropriate dual specificity.The chelators and metal chelate complexes used are often radioactive,using radionuclides such as cobalt-57 (Goodwin et al., U.S. Pat. No.4,863,713), indium-111 (Barbet et al., U.S. Pat. No. 5,256,395 and U.S.Pat. No. 5,274,076, Goodwin et al., J. Nucl. Med. 33:1366-1372 (1992),and Kranenborg et al. Cancer Res (suppl.) 55:5864s-5867s (1995) andCancer (suppl.) 80:2390-2397 (1997)) and gallium-68 (Boden et al.,Bioconjugate Chem. 6:373-379, (1995) and Schuhmacher et al. Cancer Res.55:115-123 (1995)) for radioimmuno-imaging. Because the Abs were raisedagainst the chelators and metal chelate complexes, they have remarkablespecificity for the complex against which they were originally raised.Indeed, the bsAbs of Boden et al. have specificity for singleenantiomers of enantiomeric mixtures of chelators and metal-chelatecomplexes. This great specificity has proven to be a disadvantage in onerespect, in that other nuclides such as yttrium-90 and bismuth-213,useful for radioimmunotherapy (RAIT), and gadolinium, useful for MRI,cannot be readily substituted into available reagents for alternativeuses. As a result, iodine-131, a non-metal, has been adopted for RAITpurposes by using an I-131-labeled indium-metal-chelate complex in thesecond targeting step. A second disadvantage to this methodologyrequires that antibodies be raised against every agent desired fordiagnostic or therapeutic use.

Thus, there is a continuing need for an immunological agent which can bedirected to diseased tissue and is reactive with a subsequentlyadministered linker moiety which is bonded to or associated with atherapeutic or diagnostic/detection metal chelate complex or atherapeutic or diagnostic/detection chelator.

The present invention relates to recombinantly produced chimeric,humanized and human monoclonal antibodies directed against cancers,including colorectal, pancreatic, and ovarian cancers. Chimeric,humanized and human monoclonal antibodies cause less production of humananti-mouse antibodies than completely murine antibodies. Additionally,when the antibodies are covalently conjugated to a diagnostic ortherapeutic reagent, they retain their binding characteristics. Further,if the human, humanized or chimeric antibodies have human constantregions that can be immunologically functional in patients, such as isthe case for IgG₁, then these can also be active against such tumors asnaked, or unconjugated, antibodies, and as such may also potentiate theantitumor effects of other therapeutic modalities, such as chemotherapyand radiation.

Colorectal, pancreatic, and ovarian cancers remain importantcontributors to cancer mortality. Their response to traditionalchemotherapy and radiation therapy is mixed, however. Furthermore, theseconventional forms of therapy have toxic side effects that limit theirutility.

The use of monoclonal antibodies offers an alternative to traditionalchemotherapy and radiation therapy. Tumor-specific and tumor-associatedmonoclonal antibodies can function alone (naked antibody therapy) or asconjugates in treatment regimens. The use of targeting monoclonalantibodies conjugated to radionuclides or other cytotoxic agents offersthe possibility of delivering such agents directly to the tumor site,thereby limiting the exposure of normal tissues to toxic agents(Goldenberg, Semin. Nucl. Med., 19: 332 (1989); Goldenberg, D M,Radioimmunotherapy, in: Nuclear Medicine Annual 2001, L. Freeman, ed.,Lippincott, William & Wilkins, Philadelphia, 2001, pp. 167-206). Inrecent years, the potential of antibody-based therapy and its accuracyin the localization of tumor-associated antigens have been demonstratedboth in the laboratory and clinical studies (see, e.g., Thorpe, TIBTECH,11: 42 (1993); Goldenberg, Scientific American, Science & Medicine, 1:64 (1994); Baldwin et al., U.S. Pat. Nos. 4,923,922 and 4,916,213;Young, U.S. Pat. No. 4,918,163; U.S. Pat. No. 5,204,095; Irie et al.,U.S. Pat. No. 5,196,337; Hellstrom et al., U.S. Pat. Nos. 5,134,075 and5,171,665, Thorpe et al., U.S. Pat. No. 6,342,221, and Epstein et al.,U.S. Pat. Nos. 5,965,132, 6,004554, 6,071,491, 6,017,514, 5,882,626 and5,019,368. In general, the use of radiolabeled antibodies or antibodyfragments against tumor-associated markers for localization of tumorshas been more successful than for therapy, in part because antibodyuptake by the tumor is generally low, ranging from only 0.01% to 0.001%of the total dose injected (Vaughan et al., Brit. J. Radiol., 60: 567(1987)). Increasing the concentration of the radiolabel to increase thedosage to the tumor is generally counterproductive, as this alsoincreases exposure of healthy tissue to radioactivity.

Mu-9 is a murine monoclonal antibody of the IgG₁ subtype, directedagainst the colon-specific antigen-p mucin (CSAp). CSAp is atumor-associated antigen that is present in a high percentage ofcolorectal, as well as pancreatic and ovarian cancers. (Gold et al.,Cancer Res., 50: 6405 (1990), and references cited therein). Inpre-clinical and clinical testing, the antibody has shown excellenttumor targeting ability (Blumenthal et al., Int. J. Cancer, 22: 292(1989); Sharkey et al., Cancer, 73(suppl): 864 (1994)). Mu-9 has anadvantage over other antibodies that target tumor antigens because itrecognizes an epitope, which is not present in the circulation (Pant etal., Cancer, 50: 919 (1982)). Circulating antigen can alter the deliveryof antibody therapy because the antibody forms circulating immunecomplexes, which in turn could affect tumor targeting and antibodypharmacokinetics and biodistribution.

As with most other promising non-human antibodies, the clinical use ofmurine Mu-9 may be limited by the development in humans of anti-mouseantibody (HAMA) responses. This can limit the diagnostic/detection andtherapeutic usefulness of the antibodies, not only because of thepotential anaphylactic problem, but also because a major portion of thecirculating antibody may be complexed to and sequestered by thecirculating anti-mouse antibodies. The production of HAMA may alsoaffect the accuracy of murine antibody-based immunoassays. Thus, HAMAresponses in general pose a potential obstacle to realizing the fulldiagnostic and therapeutic potential of the Mu-9 antibody.

In order to maximize the value of the Mu-9 antibody as a therapeutic ordiagnostic/detection modality and to increase its utility in multipleand continuous administration modalities and settings, an object of thisinvention is to provide a mouse-human chimeric mAb (cMu-9), a fullyhuman, and a humanized mAb (hMu-9) that relate to Mu-9 by retaining theantigen-binding specificity of Mu-9, but that elicit reduced HAMA orother immune responses in a subject receiving the same.

Another object of this invention is to provide DNA sequences that encodethe amino acid sequences of the variable regions of the light and heavychains of the cMu-9, human Mu-9, and hMu-9 mAbs, including thecomplementarity-determining regions (CDRS).

A further object of this invention is to provide conjugates of thehMu-9, human Mu-9, and cMu-9 mAbs containing therapeutic ordiagnostic/detection modalities.

Another object of this invention is to provide combinations ofantibodies with CSAp antibody or antibodies with othercarcinoma-targeting antibodies, wherein said antibodies can be used asnaked immunoglobulins or as conjugates with drugs, toxins, isotopes,cytokines, enzymes, enzyme-inhibitors, hormones, hormone antagonists,and other therapy-enhancing moieties.

Yet another object of this invention is to provide methods of therapyand diagnosis/detection that utilize the humanized, chimeric and fullyhuman MAbs of the invention.

SUMMARY OF THE INVENTION

The present invention provides a monoclonal (MAb)-antibody or fragmentthereof that binds to a colon-specific antigen-p mucin (CSAp) antigen.Preferably, the monoclonal antibody or fragment thereof binds the Mu-9epitope. Still preferred, the monoclonal antibody or fragment thereof ishumanized, chimerized or fully human.

The invention also provides a humanized Mu-9 (hMu-9) monoclonal antibody(mAb) or a fragment thereof. The hMu-9 antibody or fragment contains thecomplementarity-determining regions (CDRs) of the light and heavy chainvariable regions of a non-human Mu-9 antibody, which are joined to theframework (FR) regions of the light and heavy chain variable regions ofa human antibody, which are subsequently joined to the light and heavychain constant regions of a human antibody. This humanized antibody orfragment retains the CSAp antigen specificity of the parental Mu-9antibody, but is less immunogenic in a human subject.

In another aspect, the invention provides a chimeric Mu-9 (cMu-9)monoclonal antibody or fragment thereof. The cMu-9 antibody or fragmentcontains the light and heavy chain variable regions of a non-human Mu-9antibody, which are joined to the light and heavy chain constant regionsof a human antibody. This chimeric antibody retains the CSAp antigenspecificity of the parental Mu-9 antibody, but is less immunogenic in ahuman subject.

Also contemplated in the present invention is a fully human Mu-9antibody and fragments thereof.

Also contemplated in the present invention is a humanized antibody orfragment thereof comprising the complementarity-determining regions(CDRs) of a murine anti-CSAp MAb and the framework (FR) regions of thelight and heavy chain variable regions of a human antibody and the lightand heavy chain constant regions of a human antibody, wherein the CDRsof the light chain variable region of the humanized anti-CSAp MAbcomprises CDR1 comprising an amino acid sequence of RSSQSIVHSNGNTYLE;CDR2 comprising an amino acid sequence of KVSNRFS and CDR3 comprising anamino acid sequence of FQGSRVPYT; and the CDRs of the heavy chainvariable region of the humanized anti-CSAp MAb comprises CDR1 comprisingan amino acid sequence of EYVIT; CDR2 comprising an amino acid sequenceof EIYPGSGSTSYNEKFK and CDR3 comprising an amino acid sequence of EDL.

The present invention further provides a CDR-grafted humanized heavychain comprising the complementarity-determining regions (CDRs) of amurine anti-CSAp MAb and the framework region of the heavy chainvariable region of a human antibody and the heavy chain constant regionof a human antibody, wherein the CDRs of the heavy chain variable regionof the humanized anti-CSAp MAb comprises CDR1 comprising an amino acidsequence of EYVIT; CDR2 comprising an amino acid sequence ofEIYPGSGSTSYNEKFK and CDR3 comprising an amino acid sequence of EDL.

In a related vein, the present invention provides a CDR-graftedhumanized light chain comprising the complementarity determining regions(CDRs) of a murine anti-CSAp MAb and the framework region of the lightchain variable region of a human antibody and the light chain constantregion of a human antibody, wherein the CDRs of the light chain variableregion of the humanized anti-CSAp MAb comprises CDR1 comprising an aminoacid sequence of RSSQSIVHSNGNTYLE; CDR2 comprising an amino acidsequence of KVSNRF and CDR3 comprising an amino acid sequence ofFQGSRVPYT.

The invention further relates to a diagnostic/detection immunoconjugatecomprising an antibody component comprising an anti-CSAp MAb or fragmentthereof or an antibody fusion protein or fragment thereof of any one ofanti-CSAp antibodies described herein, wherein the antibody component isbound to at least one diagnostic/detection agent. Preferably, thediagnostic/detection immunoconjugate comprises at least one photoactivediagnostic/detection agent. More preferably, the photoactivediagnostic/detection agent comprises a chromagen or dye. Stillpreferred, the diagnostic/detection agent is a radionuclide with anenergy between 20 and 2,000 keV.

In a related vein, the invention further provides a therapeuticimmunoconjugate comprising an antibody component comprising an anti-CSApMAb or fragment thereof or an antibody fusion protein or fragmentthereof of any one of anti-CSAp antibodies described herein, wherein theantibody component is bound to at least one therapeutic agent. In apreferred embodiment, the therapeutic agent is a radionuclide, boron,gadolinium or uranium atoms, an immunomodulator, a cytokine, a hormone,a hormone antagonist, an enzyme, an enzyme inhibitor, a photoactivetherapeutic agent, a cytotoxic drug, a toxin, an angiogenesis inhibitor,a second, different antibody, and a combination thereof. When thetherapeutic immunoconjugate is a radionuclide, the energy is preferablybetween 20 and 10,000 keV.

The invention further provides multivalent, multispecific antibody orfragment thereof comprising one or more antigen binding sites havingaffinity toward a CSAp target antigen and one or more hapten bindingsites having affinity towards hapten molecules.

The invention also relates to an antibody fusion protein or fragmentthereof comprising at least two anti-CSAp MAbs or fragments thereof, asdescribed herein. Similarly, the invention contemplates an antibodyfusion protein or fragment thereof that comprises at least one firstanti-CSAp MAb or fragment thereof, as described herein, and at least onesecond MAb or fragment thereof, other than the anti-CSAp antibodies ofthe present invention.

The invention also provides a method of treating a malignancy in asubject, comprising the step of administering to said subject atherapeutically effective amount of an anti-CSAp antibody or fragmentthereof, formulated in a pharmaceutically acceptable vehicle.

Similarly, the present invention provides for a method of treating ordiagnosing/detecting a malignancy in a subject, comprising (i)administering to a subject in need thereof the antibody or fragmentsthereof of the present invention; (ii) waiting a sufficient amount oftime for an amount of the non-binding protein to clear the subject'sbloodstream; and (iii) administering to said subject a carrier moleculecomprising a diagnostic agent, a therapeutic agent, or a combinationthereof, that binds to a binding site of the antibody.

The invention also provides for a DNA sequence comprising a nucleic acidencoding a anti-CSAp MAb or fragment thereof selected from the groupconsisting of

(a) an anti-CSAp MAb or fragment thereof as described herein;

(b) an antibody fusion protein or fragment thereof comprising at leasttwo of the MAbs or fragments thereof;

(c) an antibody fusion protein or fragment thereof comprising at leastone first anti-CSAp MAb or fragment thereof comprising said MAb orfragment thereof of any one of the anti-CSAp antibodies of the presentinvention and at least one second MAb or fragment thereof, other thanthe MAb or fragment thereof of the present invention; and

(d) an antibody fusion protein or fragment thereof comprising at leastone first MAb or fragment thereof comprising said MAb or fragmentthereof of any one of the antibodies described herein and at least onesecond MAb or fragment thereof, other than the MAb or fragment thereofof any one of the antibodies described herein, wherein said second MAbis selected from the group consisting of CEA, EGP-1, EGP-2, MUC-1,MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3,KS-1, CD40, VEGF antibody, and the antibody A33, and a combinationthereof.

The invention also relates to a method of delivering adiagnostic/detection agent, a therapeutic agent, or a combinationthereof to a target, comprising: (i) administering to a subject theantibody or fragments thereof of any one the anti-CSAp antibodies of thepresent invention; (ii) waiting a sufficient amount of time for anamount of the non-binding protein to clear the subject's blood stream;and (iii) administering to said subject a carrier molecule comprising adiagnostic/detection agent, a therapeutic agent, or a combinationthereof, that binds to a binding site of said antibody.

Described herein is also a method of treating a malignancy in a subjectcomprising administering to said subject a therapeutically effectiveamount of an antibody or fragment thereof or an antibody fusion proteinor fragment thereof comprising at least two MAbs or fragments thereof,wherein at least one anti-CSAp MAb or fragment thereof or fusionproteins or fragments thereof as described herein, formulated in apharmaceutically suitable excipient.

The present invention further relates to a method of treating a cancerin a subject comprising (i) administering to said subject atherapeutically effective amount of a composition comprising a nakedanti-CSAp MAb or fragment thereof or a naked antibody fusion protein orfragment thereof of any one of the anti-CSAp antibodies of the presentinvention (ii) formulating the naked CSAp MAb or fragment thereof orantibody fusion protein or fragment thereof in a pharmaceuticallysuitable excipient.

In an additional aspect, the invention provides conjugates in which thehMu-9, human Mu-9, or cMu-9 is bonded to a diagnostic/detection ortherapeutic reagent.

In a further aspect, the invention provides that unconjugated (naked)hMu-9, human Mu-9, or cMu-9 is administered in combination with othertraditional as well as experimental therapy modalities, such asradiation, chemotherapy and surgery, or even with conjugates involvingother, non-CSAp antibodies, and that the combination(s) may be givensimulataneously or at different times in the therapy cycle.

In still another aspect, the invention provides methods ofdiagnosing/detecting or treating a malignancy that include administeringan effective amount of the aforementioned antibodies or conjugates. Theantibodies or conjugates may be formulated in a pharmaceuticallyacceptable vehicle.

In a further aspect, the invention provides isolated polynucleotidesthat comprise DNA sequences encoding the amino acid sequences of theCDRs of the light and heavy chain variable regions of the hMu-9, humanMu-9, or cMu-9 mAbs. Similarly, the invention provides isolatedpolynucleotides that comprise DNA sequences encoding the amino acidsequence of the light and heavy chain variable regions of the hMu-9,human Mu-9, or cMu-9 mAbs.

In yet another aspect, the invention provides amino acid sequences ofthe CDRs of the light and heavy chain variable regions of a Mu-9antibody.

The present invention also seeks to provide inter alia a bi-specificantibody or antibody fragment having at least one arm that specificallybinds a targeted tissue and at least one other arm that specificallybinds a targetable conjugate that can be modified for use in a widevariety of diagnostic and therapeutic applications.

The present inventors have discovered that it is advantageous to raisebsAbs against a targetable conjugate that is capable of carrying one ormore diagnostic/detection or therapeutic agents. By utilizing thistechnique, the characteristics of the chelator, metal chelate complex,therapeutic agent or diagnostic/detection agent can be varied toaccommodate differing applications, without raising new bsAbs for eachnew application. Further, by using this approach, two or more distinctchelators, metal chelate complexes or therapeutic agents can be usedwith the inventive bsAb.

Provided in the present invention is a method of treating or identifyingdiseased tissues in a subject, comprising:

(A) administering to a subject a bi-specific antibody or antibodyfragment having at least one arm that specifically binds a targetedtissue and at least one other arm that specifically binds a targetableconjugate, wherein the arm that specifically binds a targeted tissue isa Mu-9 antibody;

(B) optionally, administering to the subject a clearing composition, andallowing said composition to clear non-localized antibodies or antibodyfragments from circulation;

(C) administering to the subject a first targetable conjugate whichcomprises a carrier portion which comprises or bears at least oneepitope recognizable by said at least one other arm of said bi-specificantibody or antibody fragment, and one or more conjugated therapeutic ordiagnostic agents; and

(D) when said therapeutic agent is an enzyme, further administering tothe subject

-   -   1) a prodrug, when said enzyme is capable of converting said        prodrug to a drug at the target site; or    -   2) a drug which is capable of being detoxified in said subject        to form an intermediate of lower toxicity, when said enzyme is        capable of reconverting said detoxified intermediate to a toxic        form, and, therefore, of increasing the toxicity of said drug at        the target site, or    -   3) a prodrug which is activated in said subject through natural        processes and is subject to detoxification by conversion to an        intermediate of lower toxicity, when said enzyme is capable of        reconverting said detoxified intermediate to a toxic form, and,        therefore, of increasing the toxicity of said drug at the target        site, or    -   4) a second targetable conjugate which comprises a carrier        portion which comprises or bears at least one epitope        recognizable by said at least one other arm of said bi-specific        antibody or antibody fragment, and a prodrug, when said enzyme        is capable of converting said prodrug to a drug at the target        site.

The invention further provides a targetable conjugate that comprises atleast two HSG haptens.

Also contemplated herein is a method for detecting or treating tumorsexpressing CSAp in a mammal, comprising:

(A) administering an effective amount of a bispecific antibody orantibody fragment comprising at least one arm that specifically binds atargeted tissue and at least one other arm that specifically binds atargetable conjugate, wherein the one arm that specifically binds atargeted tissue is a Mu-9 antibody or fragment thereof; and

(B) administering a targetable conjugate selected from the groupconsisting of

(i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;

(ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;

(iii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

This method optionally comprises administering to the subject a clearingcomposition, and allowing the composition to clear non-localizedantibodies or antibody fragments from circulation.

Further, the invention provides a kit useful for treating or identifyingdiseased tissues in a subject comprising:

(A) a bi-specific antibody or antibody fragment having at least one armthat specifically binds a targeted tissue and at least one other armthat specifically binds a targetable conjugate, wherein said one armthat specifically binds a targeted tissue is a Mu-9 antibody or fragmentthereof;

(B) a first targetable conjugate which comprises a carrier portion whichcomprises or bears at least one epitope recognizable by said at leastone other arm of said bi-specific antibody or antibody fragment, and oneor more conjugated therapeutic or diagnostic agents; and

(C) optionally, a clearing composition useful for clearing non-localizedantibodies and antibody fragments; and

(D) optionally, when the therapeutic agent conjugated to said firsttargetable conjugate is an enzyme,

-   -   1) a prodrug, when said enzyme is capable of converting said        prodrug to a drug at the target site; or    -   2) a drug which is capable of being detoxified in said subject        to form an intermediate of lower toxicity, when said enzyme is        capable of reconverting said detoxified intermediate to a toxic        form, and, therefore, of increasing the toxicity of said drug at        the target site, or    -   3) a prodrug which is activated in said subject through natural        processes and is subject to detoxification by conversion to an        intermediate of lower toxicity, when said enzyme is capable of        reconverting said detoxified intermediate to a toxic form, and,        therefore, of increasing the toxicity of said drug at the target        site, or    -   4) a second targetable conjugate which comprises a carrier        portion which comprises or bears at least one epitope        recognizable by said at least one other arm of said bi-specific        antibody or antibody fragment, and a prodrug, when said enzyme        is capable of converting said prodrug to a drug at the target        site.

As described herein, the targetable conjugate may consist of:

-   -   (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;    -   (ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;    -   (iii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

The invention also relates to a method of screening for a targetableconjugate comprising:

(A) contacting the targetable construct with a bi-specific antibody orantibody fragment having at least one arm that specifically binds atargeted tissue and at least one other arm that specifically binds saidtargetable conjugate to give a mixture, wherein the one arm thatspecifically binds a targeted tissue is a Mu-9 antibody or fragmentthereof; and

(B) optionally incubating said mixture; and

(C) analyzing said mixture.

The invention further provides a method for imaging malignant tissue orcells in a mammal expressing CSAp, comprising:

(A) administering an effective amount of a bispecific antibody orantibody fragment comprising at least one arm that specifically binds atargeted tissue and at least one other arm that specifically binds atargetable conjugate, wherein the one arm that specifically binds atargeted tissue is a Mu-9 antibody or fragment thereof; and

(B) administering a targetable conjugate selected from the groupconsisting of

-   -   (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;    -   (ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;    -   (iii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

The invention also provides a method of intraoperatively, endoscopicallyand intravascularly identifying/disclosing diseased tissues expressingCSAp in a subject, comprising the administration of a detectable amountof a CSAp-labeled antibody, preferably a fragment or subfragment,whereby the label is detected by a suitable probe or miniature camerawithin 48 hours of said labeled CSAp antibody/fragment administration,without the need of a clearing agent for non-targeted, labeled antibodyor fragment.

In a related vein, the invention provides a method of intraoperativelyidentifying/disclosing diseased tissues expressing CSAp, in a subject,comprising:

(A) administering an effective amount of a bispecific antibody orantibody fragment comprising at least one arm that specifically binds atargeted tissue expressing CSAp and at least one other arm thatspecifically binds a targetable conjugate, wherein the one arm thatspecifically binds a targeted tissue is a Mu-9 antibody or fragmentthereof; and

(B) administering a targetable conjugate selected from the groupconsisting of

-   -   (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;    -   (ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;    -   (iii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

The invention further relates to a method for the endoscopicidentification of diseased tissues expressing CSAp, in a subject,comprising:

(A) administering an effective amount of a bispecific antibody orantibody fragment comprising at least one arm that specifically binds atargeted tissue expressing CSAp and at least one other arm thatspecifically binds a targetable conjugate wherein the one arm thatspecifically binds a targeted tissue is a Mu-9 antibody or fragmentthereof; and

(B) administering a targetable conjugate selected from the groupconsisting of

-   -   (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;    -   (ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;    -   (iii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

Also provided herein is a method for the intravascular identification ofdiseased tissues expressing CSAp, in a subject, comprising:

(A) administering an effective amount of a bispecific antibody orantibody fragment comprising at least one arm that specifically binds atargeted tissue expressing CSAp and at least one other arm thatspecifically binds a targetable conjugate wherein the one arm thatspecifically binds a targeted tissue is a Mu-9 antibody or fragmentthereof; and

(B) administering a targetable conjugate selected from the groupconsisting of

-   -   (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;    -   (ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;    -   (iii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

The invention also relates to a method of detection of lesions during anendoscopic, laparoscopic, intravascular catheter, or surgical procedure,wherein the method comprises:

(a) injecting a subject who is to undergo such a procedure with abispecific antibody F(ab)₂ or F(ab′)₂ fragment, wherein the bispecificantibody or fragment has a first antibody binding site whichspecifically binds to a CSAp antigen, and has a second antibody bindingsite which specifically binds to a hapten, and permitting the antibodyfragment to accrete at target sites;

(b) optionally clearing non-targeted antibody fragments using agalactosylated anti-idiotype clearing agent if the bispecific fragmentis not largely cleared from circulation within about 24 hours ofinjection, and injecting a bivalent labeled hapten, which quicklylocalizes at the target site and clears through the kidneys;

(c) detecting the presence of the hapten by close-range detection ofelevated levels of accreted label at the target sites with detectionmeans, within 48 hours of the first injection, and conducting saidprocedure, wherein said detection is performed without the use of acontrast agent or subtraction agent.

In a preferred embodiment, the hapten is labeled with a diagnosticradioisotope, a MRI image enhancing agent or a fluorescent label.

The invention further relates to a method for close-range lesiondetection, during an operative, intravascular, laparoscopic, orendoscopic procedure, wherein the method comprises:

(a) injecting a subject to such a procedure parenterally with aneffective amount of a Mu-9 immunoconjugate or fragment thereof,

(b) conducting the procedure within 48 hours of the injection;

(c) scanning the accessed interior of the subject at close range with adetection means for detecting the presence of said labeled antibody orfragment thereof; and

(d) locating the sites of accretion of said labeled antibody or fragmentthereof by detecting elevated levels of said labeled antibody orfragment thereof at such sites with the detection means.

In the above examples of intraoperative, endoscopic and intravascularuses, the label attached to the diagnostic compound is capable of beingdetected by a suitable instrument or probe, including miniature cameras,which are made for said label detection (e.g., a gamma-detecting probewhen a gamma-emitting isotope is the diagnostic/detection conjugate)(see Goldenberg, U.S. Pat. Nos. 5,716,595, 6,096,289 and U.S.application Ser. No. 09/348,818, incorporated herein by reference intheir entirety.

These and other aspects and embodiments of the invention will becomeapparent by reference to the following specification and appendedclaims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the DNA and amino acid sequences of the murine Mu-9 heavyand light chain variable regions. FIG. 1A shows the DNA and amino acidsequences of the Mu-9VH obtained by RT-PCR. FIG. 1B shows the DNA andamino acid sequences of the functional Mu-9Vk (Vk2) obtained by cDNAscreening. Amino acid sequences encoded by the corresponding DNAsequences are given as one letter codes below the nucleotide sequence.Numbering of the nucleotide sequence is on the right side. The aminoacid residues in the CDR regions are shown in bold and underlined.Kabat's Ig molecule numbering is used for amino acid residues as shownby the numbering above the amino acid residues. The residues numberedwith a letter only are the insertion residues defined by Kabat numberingscheme and have the same preceeding digits as the previous one. Forexample, residues 82, 82A, 82B, and 82C in FIG. 1A are indicated as 82,A, B, and C, respectively.

FIG. 2 shows the DNA and amino acid sequences of the chimeric Mu-9(cMu-9) heavy and light chain variable regions expressed in Sp2/0 cells.FIG. 2A shows the DNA and amino acid sequences of the cMu-9VH. FIG. 2Bshows the double-stranded DNA and amino acid sequences of the cMu-9Vk.Amino acid sequences encoded by the corresponding DNA sequences aregiven as one letter codes. The amino acid residues in the CDR regionsare shown in bold and underlined. Numbering of the nucleotide sequenceis on the right side. The numbering of amino acids is same as that inFIG. 1. The restriction sites used for constructing the cMu-9 areunderlined and indicated.

FIG. 3 shows the alignment of the amino acid sequences of heavy andlight chain variable regions of a human antibody, Mu-9 and hMu-9. FIG.3A shows the VH amino acid sequence alignment of the human antibody EU(FR1-3) and NEWM (FR4) with Mu-9 and hMu-9 and FIG. 3B shows the Vκamino acid sequence alignment of the human antibody WOL with Mu-9 andhMu-9. Dots indicate the residues in Mu-9 that are identical to thecorresponding residues in the human antibodies. Boxed regions representthe CDR regions. Both N- and C-terminal residues (underlined) of hMu-9are fixed by the staging vectors used and not compared with the humanantibodies. Kabat's Ig molecule number scheme is used to number theresidues as in FIG. 1.

FIG. 4 shows the DNA and amino acid sequences of the humanized Mu-9(hMu-9) heavy and light chain variable regions expressed in Sp2/0 cells.FIG. 4A shows the DNA and amino acid sequences of the hMu-9VH and FIG.4B shows the DNA and amino acid sequences of the hMu-9Vκ. Numbering ofthe nucleotide sequence is on the right side. Amino acid sequencesencoded by the corresponding DNA sequences are given as one lettercodes. The amino acid residues in the CDR regions are shown in bold andunderlined. Kabat's Ig molecule numbering scheme is used for amino acidresidues as in FIG. 1A and FIG. 1B.

FIG. 5 shows a comparison of mMu-9 and hMu-9 in competitive bindingassays. Varying concentrations of competing Abs were used to competewith the binding of a constant amount of HRP-mMu-9 to the antigen coatedwells. hMu-9 showed comparable blocking activity as that of mMu-9. Acomparison of mMu-9 and cMu-9 can be found in in the publication byKrishnan et al. (Cancer, 80:2667-2674 (1997)).

DETAILED DESCRIPTION I. Overview

The present invention encompasses antibodies and antibody fragments. Theantibody fragments are antigen binding portions of an antibody, such asF(ab′)₂, F(ab)₂, Fab′, Fab, and the like. The antibody fragments bind tothe same antigen that is recognized by the intact antibody. For example,an anti-CSAp monoclonal antibody fragment binds to an epitope of CSAp.

The term “antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments, “Fv” fragments, consisting of the variable regionsof the heavy and light chains, recombinant single chain polypeptidemolecules in which light and heavy chain variable regions are connectedby a peptide linker (“sFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the “hypervariableregion.” Three of these so-called “hypervariable” regions or“complementarity-determining regions” (CDR) are found in each variableregion of the light or heavy chain. Each CDR is flanked by relativelyconserved framework regions (FR). The FR are thought to maintain thestructural integrity of the variable region. The CDRs of a light chainand the CDRs of a corresponding heavy chain form the antigen-bindingsite. The “hypervariability” of the CDRs accounts for the diversity ofspecificity of antibodies.

Also contemplated in the present invention are human, chimeric andhumanized anti-CSAp antibodies and fragments thereof. The humananti-CSAp antibody of the present invention is preferably against theMu-9 antigen. A fully human antibody is an antibody obtained, forexample, from transgenic mice that have been “engineered” to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A fully human antibody also can be constructed bygenetic or chromosomal transfection methods, as well as phage displaytechnology, all of which are known in the art. See, for example,McCafferty et al., Nature 348:552-553 (1990) for the production of humanantibodies and fragments thereof in vitro, from immunoglobulin variabledomain gene repertoires from unimmunized donors. In this technique,antibody variable domain genes are cloned in-frame into either a majoror minor coat protein gene of a filamentous bacteriophage, and displayedas functional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. In this way, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats, for their review, see e.g. Johnson and Chiswell, CurrentOpinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated intheir entirety by reference.

The present invention also provides a chimeric anti-CSAp monoclonalantibody or fragment thereof. The chimeric anti-CSAp antibody orfragment contains a light and heavy chain variable region of a non-humananti-CSAp antibody, which are joined to the light and heavy chainconstant regions of a human antibody. Preferably, the light and heavychain variable regions come from a murine anti-CSAp antibody. In apreferred embodiment, the anti-CSAp antibody binds a Mu-9 epitope on theCSAp antigen. Accordingly, a Mu-9 antibody is an anti-CSAp antibody thatbinds to the Mu-9 epitope.

The process for making cMu-9 is described in detail below. Briefly,cDNAs encoding the Vκ and V_(H) regions of the Mu-9 mAb have beenisolated and separately recombinantly subcloned into mammalianexpression vectors that contain the genes a human K light chain constantregion sequence and a human γ1 chain sequence, respectively.Cotransfection of mammalian cells with these two recombinant DNAsresulted in expression of a cMu-9 mAb that, like the parent Mu-9 mAb,bound avidly to the CSAp antigen.

In a preferred embodiment, the light chain variable region of the cMu-9antibody comprises the amino acids of SEQ. ID NO: (FIG. 2B) or the heavychain variable region of the cMu-9 antibody comprises the amino acids ofSEQ. ID NO: (FIG. 2A). Still preferred, the light chain variable regionof the cMu-9 antibody comprises the amino acids of SEQ. ID NO: (FIG. 2B)and the heavy chain variable region of the cMu-9 antibody comprises theamino acids of SEQ. ID NO: (FIG. 2A).

The present invention further provides a humanized Mu-9 (hMu-9)monoclonal antibody (mAb) or a fragment thereof. The hMu-9 antibody orfragment contains the complementarity-determining regions (CDRs) of thelight and heavy chain variable regions of a non-human Mu-9 antibody,which are joined to the framework (FR) regions of the light and heavychain variable regions of a human antibody, which are subsequentlyjoined to the light and heavy chain constant regions of a humanantibody. This humanized antibody or fragment retains the CSAp antigenspecificity of the parental Mu-9 antibody, but is less immunogenic in ahuman subject.

Methods for making hMu-9 are described in detail below. Briefly,however, to make hMu-9, the CDRs of the Vκ and V_(H) DNAs have beenrecombinantly linked to the framework (FR) sequences of the human Vκ andV_(H) regions, respectively, which are subsequently linked,respectively, to the human kappa and γ1 constant regions, so as toexpress in mammalian cells as described above hMu-9.

In another embodiment of the present invention, hMu-9, the CDRs of thelight chain variable region comprise CDR1 comprising amino acids 24 to34 of SEQ ID NO: (FIG. 2B), CDR2 comprising amino acids 50 to 56 of SEQID NO: (FIG. 2B), and CDR3 comprising amino acids 89 to 97 of SEQ ID NO:(FIG. 2B); and the CDRs of the heavy chain variable region comprise CDR1comprising amino acids 31 to 35 of SEQ ID NO: (FIG. 2A), CDR2 comprisingamino acids 50 to 64 of SEQ ID NO: (FIG. 2A), and CDR3 comprising aminoacids 95 to 97 of SEQ ID NO: (FIG. 2A).

Other preferred embodiments of the invention include anti-CSAp antibodyfragments comprising the light chain variable region of SEQ ID NO: (FIG.4B) and/or the heavy chain variable region of SEQ ID NO: (FIG. 4A).

In this specification, the expressions “cMu-9” or “cMu-9 mAb” areintended to refer to the chimeric monoclonal antibody constructed byjoining or subcloning the non-human Vk and VH regions to the humanconstant light and heavy chains, respectively. The expressions “hMu-9”or “hMu-9 mAb” are intended to refer to the humanization of the chimericmonoclonal antibody by replacing the non-human FR sequences in cMu-9with that of human framework regions. Preferably, the anti-CSAphumanized antibodies and fragments thereof of the present inventioncomprise framework region sequences where at least one amino acid of thecorresponding non-human light or heavy chain framework regions isretained. Preferably, an amino acid from the murine antibody FR isretained in the same position of the corresponding humanized antibody.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species, such as acat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, is transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule is derived from those of a human antibody.

The present invention also contemplates anti-CSAp antibody fragments.The antibody fragments are antigen binding portions of an antibody, suchas F(ab′)₂, F(ab)₂, Fab′, Fab, and the like. The antibody fragmentscontain one or more CDRs of the intact antibody and bind to the sameantigen that is recognized by the intact antibody. For example, ananti-colon-specific antigen-p (CSAp) monoclonal antibody fragment bindsto an epitope of colon-specific antigen-p.

Also, the present invention provides a bi-specific antibody or antibodyfragment having at least one arm that is reactive against a targetedtissue and at least one other arm that is reactive against a targetableconstruct. The targetable construct is comprised of a carrier portionand at least 2 units of a recognizable hapten. Examples of recognizablehaptens include, but are not limited to, histamine succinyl glycine(HSG) and fluorescein isothiocyanate. The targetable construct may beconjugated to a variety of agents useful for treating or identifyingdiseased tissue. Examples of conjugated agents include, but are notlimited to, chelators, metal chelate complexes, drugs, toxins (e.g.,ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonasexotoxin, Pseudomonas endotoxin) and other effector molecules.Additionally, enzymes useful for activating a prodrug or increasing thetarget-specific toxicity of a drug can be conjugated to the targetableconstruct. Thus, the use of bsAb which are reactive to a targetableconstruct allows a variety of therapeutic and diagnostic/detectionapplications to be performed without raising new bsAb for eachapplication.

Additionally, the present invention encompasses a method for detectingor treating target diseased cells or tissues in a mammal, comprisingadministering an effective amount of a bispecific antibody or antibodyfragment comprising at least one arm that specifically binds a targetedtissue and at least one other arm that specifically binds a targetableconjugate.

Antibodies that do not target the CSAp antigen can be used in thisinvention. For example, antibodies against other antigens associatedwith carcinomas, particularly carcinomas of the gastrointestinal system(colon, rectum, pancreas tumors) and ovarian cancer, can be combinedwith CSAp antibodies and also used as fusion partners with CSApantibodies. Antibodies against intracellular and other antigensassociated with necrosis, angiogenesis factors, immune response factors(e.g., CD40), as well as products of oncogenes, may also be used incombination with CSAp antibodies and as fusion partners for CSApantibodies. Anti-necrosis antibodies are described in Epstein et al.,U.S. Pat. Nos. 6,071,491, 6,017,514, 5,019,368 and 5,882,626, and areincorporated by reference.

Immunoconjugates between chimeric, humanized and human anti-CSApantibodies or fragments thereof and a diagnostic or therapeutic reagent,formulated in pharmaceutically acceptable vehicles (see, e.g.,Remington's Pharmaceutical Sciences, 18^(th) ed., Mack Publishing Co.,Easton, Pa., 1990) can be prepared. An immunoconjugate is a conjugate ofan antibody component with a therapeutic or diagnostic agent. As random(non-specific) conjugation often results in products with reducedbinding activity, it is preferred to use conjugates in which the reagentis site-specifically bound to the antibody through, for example,carbohydrate moieties, such as through oxidized carbohydratederivatives. Carbohydrate moieties can be introduced into an antibody bysite-specific mutagenesis without altering the immunoreactivity. Methodsfor the production of such conjugates and their use in diagnostics andtherapeutics are provided, for example in Shih et al., U.S. Pat. No.5,057,313; Shih et al., Int. J. Cancer 41: 832 (1988); and Hansen etal., U.S. Pat. No. 5,443,953, the contents of which are incorporatedherein by reference. Direct linkage of the reagent to oxidizedcarbohydrate without the use of a polymeric carrier is described inMcKearn et al., U.S. Pat. No. 5,156,840, which is also incorporated byreference.

A wide variety of diagnostic/detection and therapeutic reagents can beadvantageously conjugated to the antibodies of the invention. Theseinclude, but are not limited to, different classes of chemotherapeuticagents, such as anthracyclines, antibiotics, alkylating agents,anti-mitotic agents, anti-angiogenesis agents, plant alkaloids,COX-inhibitors, antimetabolites (e.g., methotrexate), doxorubicin,CPT-11, oxaliplatin, taxol and other taxanes, and the like; chelators,such as DTPA, to which detectable labels such as fluorescent moleculesor cytotoxic agents, such as heavy metals or radionuclides can becomplexed; and toxins such as Pseudomonas exotoxin, RNAse, gelonin, andthe like.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,or a subfragment, and is useful in the treatment of a disease. Examplesof therapeutic agents include antibodies, antibody fragments, drugs,toxins, enzymes, enzyme-inhibitors, nucleases, hormones, hormoneantagonists, immunomodulators, chelators, boron compounds, uraniumatoms, photoactive agents or dyes and radionuclides. Radionuclides intherapeutic agents, which substantially decay by beta-particle emissioninclude, but are not limited to: P-32, P-33, Sc47, Fe-59, Cu-64, Cu-67,Se-75, As-77, Sr-89, Y-90, Mo-99, Rh-105, Pd-109, Ag-111, I-125, I-131,Pr-142, Pr-143, Pm-149, Sm-153, Th-161, Ho-166, Er-169, Lu-177, Re-186,Re-188, Re-189, Ir-194, Au-198, Au-199, Pb-211, Pb-212, and Bi-213.Maximum decay energies of useful beta-particle-emitting nuclides arepreferably 20-5,000 keV, more preferably 100-4,000 keV, and mostpreferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161,Os-189m and Ir-192. Decay energies of useful Auger-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV.

Enzymes are also useful therapeutic agents. For example, alkalinephosphatase for use in combination with phosphate-containing prodrugs(U.S. Pat. No. 4,975,278); arylsulfatase for use in combination withsulfate-containing prodrugs (U.S. Pat. No. 5,270,196); peptidases andproteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidase (U.S. Pat. Nos. 5,660,829; 5,587,161; 5,405,990) andcathepsins (including cathepsin B and L), for use in combination withpeptide-based prodrugs; D-alanylcarboxypeptidases for use in combinationwith D-amino acid-modified prodrugs; carbohydrate-cleaving enzymes suchas beta-galactosidase and neuraminidase for use in combination withglycosylated prodrugs (U.S. Pat. Nos. 5,561,119; 5,646,298);.beta.-lactamase for use in combination with beta-lactam-containingprodrugs; penicillin amidases, such as penicillin-V-amidase (U.S. Pat.No. 4,975,278) or penicillin-G-amidase, for use in combination withdrugs derivatized at their amino nitrogens with phenoxyacetamide orphenylacetamide groups; and cytosine deaminase (U.S. Pat. Nos.5,338,678; 5,545,548) for use in combination with 5-fluorocytosine-basedprodrugs (U.S. Pat. No. 4,975,278), are suitable therapeutic agents forthe present invention.

Anti-angiogenic agents (or angiogenesis inhibitors) suitable for use incombination therapy or for conjugating to antibodies includeangiostatin, endostatin, vasculostatin, canstatin and maspin.

Other useful therapeutic agents include metals, such as those as part ofa photodynamic therapy, and nuclides, such as those valuable intherapies based on neutron capture procedures. Specifically, zinc,aluminum, gallium, lutetium and palladium are useful for photodynamictherapy and B-10, Gd-157 and U-235 are useful for neutron capturetherapy.

A diagnostic/detection agent is a molecule or atom which is administeredconjugated to an antibody moiety, i.e., antibody or antibody fragment,or subfragment, and is useful in diagnosing/detecting a disease bylocating the cells containing the disease-associated antigen. Usefuldiagnostic/detection agents include, but are not limited to,radioisotopes, dyes (such as with the biotin-streptavidin complex),radiopaque materials (e.g., iodine, barium, gallium, and thalliumcompounds and the like), contrast agents, fluorescent compounds ormolecules and enhancing agents (e.g., paramagnetic ions) for magneticresonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI techniqueand the preparation of antibodies conjugated to a MRI enhancing agentand is incorporated in its entirety by reference. Preferably, thediagnostic/detection agents are selected from the group consisting ofradioisotopes for nuclear imaging, intraoperative and endoscopicdetection; enhancing agents for use in magnetic resonance imaging or inultrasonography; radiopaque and contrast agents for X-rays and computedtomography; and fluorescent compounds for fluoroscopy, includingendoscopic fluoroscopy. Fluorescent and radioactive agents conjugated toantibodies or used in bispecific, pretargeting methods, are particularlyuseful for endoscopic, intraoperative or intravascular detection of thetargeted antigens associated with diseased tissues or clusters of cells,such as malignant tumors, as disclosed in Goldenberg U.S. Pat. Nos.5,716,595, 6,096,289 and U.S. application Ser. No. 09/348,818,incorporated herein by reference in their entirety, particularly withgamma-, beta-, and positron-emitters. Radionuclides useful for positronemission tomography include, but are not limited to: F-18, Mn-51,Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68, As-72, Br-75, Br-76, Rb-82m,Sr-83, Y-86, Zr-89, Tc-94m, In-110, I-120, and I-124. Total decayenergies of useful positron-emitting radionuclides are preferably <2,000keV, more preferably under 1,000 keV, and most preferably <700 keV.Radionuclides useful as diagnostic agents utilizing gamma-ray detectioninclude, but are not limited to: Cr-51, Co-57, Co-58, Fe-59, Cu-67,Ga-67, Se-75, Ru-97, Tc-99m, In-111, In-114m, I-123, I-125, I-131,Yb-169, Hg-197, and Tl-201. Decay energies of useful gamma-ray emittingradionuclides are preferably 20-2000 keV, more preferably 60-600 keV,and most preferably 100-300 keV.

Paramagnetic ions suitable for the present invention include includechromium (III), manganese (II), iron (III), iron (II), cobalt (II),nickel (II), copper (II), neodymium (III), samarium (all), ytterbium(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) and erbium (III), with gadolinium being particularlypreferred.

Ions useful in other contexts, such as X-ray imaging, include but arenot limited to lanthanum (III), gold (III), lead (II), and especiallybismuth (III). Fluorescent labels include rhodamine, fluorescein andrenographin. Rhodamine and fluorescein are often linked via anisothiocyanate intermediate.

Metals are also useful in diagnostic agents, including those formagnetic resonance imaging techniques. These metals include, but are notlimited to: Gadolinium, manganese, iron, chromium, copper, cobalt,nickel, dysprosium, rhenium, europium, terbium, holmium and neodymium.In order to load an antibody component with radioactive metals orparamagnetic ions, it may be necessary to react it with a reagent havinga long tail to which are attached a multiplicity of chelating groups forbinding the ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chain havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose. Chelates are coupled to the peptide antigens usingstandard chemistries. The chelate is normally linked to the antibody bya group which enables formation of a bond to the molecule with minimalloss of immunoreactivity and minimal aggregation and/or internalcross-linking. Other, more unusual, methods and reagents for conjugatingchelates to antibodies are disclosed in U.S. Pat. No. 4,824,659 toHawthorne, entitled “Antibody Conjugates,” issued Apr. 25, 1989, thedisclosure of which is incorporated herein in its entirety by reference.Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with diagnostic isotopes inthe general energy range of 20 to 2,000 keV. The same chelates, whencomplexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI, when used along with the antibodies ofthe invention. Macrocyclic chelates such as NOTA, DOTA, and TETA are ofuse with a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates such asmacrocyclic polyethers, which are of interest for stably bindingnuclides, such as ²²³Ra for RAIT are encompassed by the invention.

Radiopaque and contrast materials are used for enhancing X-rays andcomputed tomography, and include iodine compounds, barium compounds,gallium compounds, thallium compounds, etc. Specific compounds includebarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexyl, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to humans andother primatesbovines (e.g, cattle), ovines (e.g., sheep), caprines(e.g., goats), porcines (e.g., swine), equines (e.g., horses), canines(e.g., dogs), felines (e.g., cats), etc. It is not intended that theterm be limited to a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are encompassed bythe term.

II. Preparation of Antibodies

Monoclonal antibodies (MAbs) are a homogeneous population of antibodiesto a particular antigen and the antibody comprises only one type ofantigen binding site and binds to only one epitope on an antigenicdeterminant. Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art. See, for example,Kohler and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.),CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991) [hereinafter “Coligan”]. Briefly, monoclonal antibodies canbe obtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS 1N MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Abs to peptide backbones are generated by well-known methods for Abproduction. For example, injection of an immunogen, such as(peptide)_(n)-KLH, wherein KLH is keyhole limpet hemocyanin, and n=1-30,in complete Freund's adjuvant, followed by two subsequent injections ofthe same immunogen suspended in incomplete Freund's adjuvant intoimmunocompetent animals, is followed three days after an i.v. boost ofantigen, by spleen cell harvesting. Harvested spleen cells are thenfused with Sp2/0-Ag14 myeloma cells and culture supernatants of theresulting clones analyzed for anti-peptide reactivity using adirect-binding ELISA. Fine specificity of generated Abs can be analyzedfor by using peptide fragments of the original immunogen. Thesefragments can be prepared readily using an automated peptidesynthesizer. For Ab production, enzyme-deficient hybridomas are isolatedto enable selection of fused cell lines. This technique also can be usedto raise antibodies to one or more of the chelates comprising thelinker, e.g., In(III)-DTPA chelates. Monoclonal mouse antibodies to anIn(III)-di-DTPA are known (Barbet '395 supra).

The antibodies used in the present invention are specific to a varietyof cell surface or intracellular tumor-associated antigens as markersubstances. These markers may be substances produced by the tumor or maybe substances which accumulate at a tumor site, on tumor cell surfacesor within tumor cells, whether in the cytoplasm or in various organellesor sub-cellular structures, or even as part of the endothelium ofvessels nourishing tumors or elaborated by the tumor vasculature. Amongsuch tumor-associated markers are those disclosed by Herberman,“Immunodiagnosis of Cancer,” in Fleisher ed., “The Clinical Biochemistryof Cancer,” page 347 (American Association of Clinical Chemists, 1979)and in U.S. Pat. Nos. 4,150,149; 4,361,544; and 4,444,744.

Tumor-associated markers have been categorized by Herberman, supra, in anumber of categories including oncofetal antigens, placental antigens,oncogenic or tumor virus associated antigens, tissue associatedantigens, organ associated antigens, ectopic hormones and normalantigens or variants thereof. Occasionally, a sub-unit of atumor-associated marker is advantageously used to raise antibodieshaving higher tumor-specificity, e.g., the beta-subunit of humanchorionic gonadotropin (HCG) or the gamma region of carcino embryonicantigen (CEA), which stimulate the production of antibodies having agreatly reduced cross-reactivity to non-tumor substances as disclosed inU.S. Pat. Nos. 4,361,644 and 4,444,744.

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. For example,humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and then,substituting human residues in the framework regions of the murinecounterparts. In a preferred embodiment, some human residues in theframework regions of the humanized anti-CSAp antibody or fragmentsthereof are replaced by their murine counterparts. It is also preferredthat a combination of framework sequences from 2 different humanantibodies are used for V_(H). Still preferred, the two human antibodiesare EU and NEWM. The constant domains of the antibody molecule isderived from those of a human antibody. The use of antibody componentsderived from humanized monoclonal antibodies obviates potential problemsassociated with the immunogenicity of murine constant regions.

A human antibody can be recovered from a transgenic mouse possessinghuman immunoglobulin loci. The mouse humoral immune system is humanizedby inactivating the endogenous immunoglobulin genes and introducinghuman immunoglobulin loci. The human immunoglobulin loci are exceedinglycomplex and comprise a large number of discrete segments which togetheroccupy almost 0.2% of the human genome. To ensure that transgenic miceare capable of producing adequate repertoires of antibodies, largeportions of human heavy- and light-chain loci must be introduced intothe mouse genome. This is accomplished in a stepwise process beginningwith the formation of yeast artificial chromosomes (YACs) containingeither human heavy- or light-chain immunoglobulin loci in germlineconfiguration. Since each insert is approximately 1 Mb in size, YACconstruction requires homologous recombination of overlapping fragmentsof the immunoglobulin loci. The two YACs, one containing the heavy-chainloci and one containing the light-chain loci, are introduced separatelyinto mice via fusion of YAC-containing yeast spheroblasts with mouseembryonic stem cells. Embryonic stem cell clones are then microinjectedinto mouse blastocysts. Resulting chimeric males are screened for theirability to transmit the YAC through their germline and are bred withmice deficient in murine antibody production. Breeding the twotransgenic strains, one containing the human heavy-chain loci and theother containing the human light-chain loci, creates progeny whichproduce human antibodies in response to immunization.

Unrearranged human immunoglobulin genes also can be introduced intomouse embryonic stem cells via microcell-mediated chromosome transfer(MMCT). See, e.g., Tomizuka et al., Nature Genetics, 16: 133 (1997). Inthis methodology microcells containing human chromosomes are fused withmouse embryonic stem cells. Transferred chromosomes are stably retained,and adult chimeras exhibit proper tissue-specific expression.

As an alternative, an antibody or antibody fragment of the presentinvention may be derived from human antibody fragments isolated from acombinatorial immunoglobulin library. See, e.g., Barbas et al., METHODS:A Companion to Methods in Enzymology 2: 119 (1991), and Winter et al.,Ann. Rev. Immunol. 12: 433 (1994), which are incorporated by reference.Many of the difficulties associated with generating monoclonalantibodies by B-cell immortalization can be overcome by engineering andexpressing antibody fragments in E. coli, using phage display. To ensurethe recovery of high affinity, monoclonal antibodies a combinatorialimmunoglobulin library must contain a large repertoire size. A typicalstrategy utilizes mRNA obtained from lymphocytes or spleen cells ofimmunized mice to synthesize cDNA using reverse transcriptase. Theheavy- and light-chain genes are amplified separately by PCR and ligatedinto phage cloning vectors. Two different libraries are produced, onecontaining the heavy-chain genes and one containing the light-chaingenes. Phage DNA is isolated from each library, and the heavy- andlight-chain sequences are ligated together and packaged to form acombinatorial library. Each phage contains a random pair of heavy- andlight-chain cDNAs and upon infection of E. coli directs the expressionof the antibody chains in infected cells. To identify an antibody thatrecognizes the antigen of interest, the phage library is plated, and theantibody molecules present in the plaques are transferred to filters.The filters are incubated with radioactively labeled antigen and thenwashed to remove excess unbound ligand. A radioactive spot on theautoradiogram identifies a plaque that contains an antibody that bindsthe antigen. Cloning and expression vectors that are useful forproducing a human immunoglobulin phage library can be obtained, forexample, from STRATAGENE Cloning Systems (La Jolla, Calif.).

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86: 3833 (1989), which is incorporated by referencein its entirety. Techniques for constructing chimeric antibodies arewell known to those of skill in the art. As an example, Leung et al.,Hybridoma 13:469 (1994), describe how they produced an LL2 chimera bycombining DNA sequences encoding the V_(κ) and V_(H) domains of LL2monoclonal antibody, an anti-CD22 antibody, with respective human κ andIgG₁ constant region domains. This publication also provides thenucleotide sequences of the LL2 light and heavy chain variable regions,V_(κ) and V_(H), respectively. Techniques for producing humanized MAbsare described, for example, by Jones et al., Nature 321: 522 (1986),Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992),Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun.150: 2844 (1993), each of which is hereby incorporated by reference.

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody can also be humanized by replacing thesequences of the murine FR in the variable domains of the chimeric MAbwith one or more different human FR. Specifically, mouse CDRs aretransferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. See, for example, Tempest et al., Biotechnology 9:266(1991) and Verhoeyen et al., Science 239: 1534 (1988). Further, theaffinity of humanized, chimeric and human MAbs to a specific epitope canbe increased by mutagenesis of the CDRs, so that a lower dose ofantibody may be as effective as a higher dose of a lower affinity MAbprior to mutagenesis. See for example, WO0029584A1

Another method for producing the antibodies of the present invention isby production in the milk of transgenic livestock. See, e.g., Colman,A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690,both of which are incorporated in their entirety by reference. Two DNAconstructs are prepared which contain, respectively, DNA segmentsencoding paired immunoglobulin heavy and light chains. The DNA segmentsare cloned into expression vectors which contain a promoter sequencethat is preferentially expressed in mammary epithelial cells. Examplesinclude, but are not limited to, promoters from rabbit, cow and sheepcasein genes, the cow α-lactoglobulin gene, the sheep β-lactoglobulingene and the mouse whey acid protein gene. Preferably, the insertedfragment is flanked on its 3′ side by cognate genomic sequences from amammary-specific gene. This provides a polyadenylation site andtranscript-stabilizing sequences. The expression cassettes arecoinjected into the pronuclei of fertilized, mammalian eggs, which arethen implanted into the uterus of a recipient female and allowed togestate. After birth, the progeny are screened for the presence of bothtransgenes by Southern analysis. In order for the antibody to bepresent, both heavy and light chain genes must be expressed concurrentlyin the same cell. Milk from transgenic females is analyzed for thepresence and functionality of the antibody or antibody fragment usingstandard immunological methods known in the art. The antibody can bepurified from the milk using standard methods known in the art.

Preparation of Chimeric, Humanized and Human Anti-CSAp Antibodies

Also contemplated in the present invention are human, humanized andchimeric anti-CSAp monoclonal antibodies used in combination with otherhuman or reengineered (e.g., chimerized, humanized) antibodies, such ascarcinoma associated antibodies including those expressed by colorectal,pancreatic and ovarian carcinomas. In a preferred embodiment, antibodiesagainst CEA, MUC1, MUC 2, MUC3, MUC 4, PAM-4, KC4, BrE3, Le-Y (e.g., B3antibody), EGFR, EGP-1, RS5 (GA733 antigen target, such as forantibodies to EGP-2, 17-1A, KS1-4, Ep-CAM), TAG-72, the A33antibody-determinant, KS-1, A3 and HER2/neu are used for combinationtherapy with humanized, chimeric or human anti-CSAp antibodies. See,e.g., Mendez et al., Nature Genetics, 15: 146-156 (1997); U.S. Pat. No.5,633,425, which are incorporated in their entirety by reference. TheBrE3 antibody is described in Couto, J, Christian, R, Peterson, J, andCeriani, R., Cancer Res. 1995; 55 (Suppl.23):5973s-5977s. The EGP-1antibody is described in U.S. Provisional Application No. 60/360,229,some of the EGP-2 antibodies are cited in Birbenet et al., in Staib etal.; and Schwartzberg in the references cited at the end of thisapplication. The KS-1 antibody is cited in Koda et al.; the A33 antibodyis cited in Ritter et al. at end; Le(y) antibody B3 described in DiCarlo et al; A3 antibody is described in Tordsson et al., all listed inthe references cited at the end of this application. Preferably,antibodies against marker antigens or receptors of gastrointestinal andovarian carcinomas are well suited for use in combination with CSApantibodies, and in particular with the Mu-9 antibodies. In a preferredembodiment, a gastrointestinal cancer is a colorectal cancer.

Also of use are antibodies against markers or products of oncogenes, orantibodies against angiogenesis factors, such as VEGF. VEGF antibodiesare described in Thorpe et al., U.S. Pat. Nos. 6,342,221, 5,965,132 and6,004,554, and are incorporated by reference in their entirety.Antibodies against certain immune response modulators, such asantibodies to CD40, are described in Todryk et al. and Turner et al.,listed in the references cited at the end of this application. Otherantibodies suitable for combination therapy include anti-necrosisantibodies as described in Epstein et al. (infra).

Cell lines and culture media used in the present invention include Mu-9hybridoma cells and Sp2/0-Ag14 myeloma cells (ATCC, Rockville, Md.). Themonoclonal hybridoma producing Mu-9 was obtained by fusing the spleenfrom a mouse that had been immunized with colon-specific antigen-p(CSAp) with SP2/0Ag14. These cells may be cultured in Hybridomaserum-free media (HSFM) (life Technologies, Grand Island, N.Y.)supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories,Logan, Utah) and antibiotics (complete media). Alternatively, they maybe cultured in Dulbecco's modified Eagle's Medium (DMEM) supplementedwith 10% FCS (Gibco/BRL, Gaithersburg, Mass.) containing 10% of FCS and75 μg/ml gantamicin (complete HSFM) or, where indicated, in HSFMcontaining only antibiotics. Selection of the transfectomas may becarried out in complete HSFM containing 500 units/ml of hygromycin(Calbiochem, San Diego, Calif.). All cell lines are preferablymaintained at 37° C. in 5% CO₂.

Obtaining V_(κ) and V_(H) Gene Segments

Isolation of the Vκ and V_(H) gene segments can be accomplished byseveral means that are well-known in the art. Two such means include,but are not limited to, PCR cloning and cDNA library screening.

PCR cloning techniques are well-known in the art. In brief, however, PCRcloning of Vκ and V_(H) gene fragments may be accomplished as follows.Poly A mRNA may be isolated from a Mu-9 hybridoma cell line usingcommercially available kits such as the Fast Track mR-NA Isolation kit(Invitrogen, San Diego, Calif.). The first strand cDNA may then bereverse transcribed from poly A mRNA using a cDNA cycle kit(Invitrogen). In this process, poly A mRNA is annealed to a murine IgGCH1-specific primer or a murine Ck-specific primer. Examples of suchprimers include CHIB (5′-ACA GTC ACT GAG CTG G-3′) and Ck3-BH1 (5′-GCCGGA TCC TGA CTG GAT GGT GGG AAG ATG GAT ACA-3′), respectively. The firststrand cDNA may be used as templates to amplify the V_(H) and Vκsequences by PCR, as described by Orlandi et al. For the Vκ region, aprimer pair such as VK1 Back (5′-GAC ATT CAG CTG ACC CAG TCT CCA-3′) andIgGKC3′ (5′-CTC ACT GGA TGG TGG GAA GAT GGA TAC AGT TGG-3′) may be used.For the V_(H) region, a primer pair such as VH1Back (5′-AGG T(C/G)(A/C)A(A/G)C TGC AG(C/G) AGT C(A/T)G G-3′) and CH1B may be used. Afteramplification, the Vκ and V_(H) fragments may then be gel-purified andcloned into a cloning vector such as the TA cloning vector (Invitrogen)for sequence analyses by the dideoxytermination method. Sequencesconfirmed to be of immunoglobulin origin may then be used to constructchimeric expression vectors using methods described by Leung et al.

As a preferred alternative to isolating the Vκ and V_(H) gene segmentsby PCR cloning, cDNA library screening may be utilized. cDNA screeningmethods also are well known in the art. In brief, however, a cDNAlibrary may be constructed from the mRNA extracted from the murine Mu-9hybridoma cells in pSPORT vector (Life Technologies). The first strandcDNA may be synthesized by priming ply A RNA from Mu-9 hybridoma with anoligo dT primer-NotI adaptor (Life Technologies). After the secondstrand synthesis and attachment of SalI adaptors, the cDNA pool may besize fractionated through a cDNA size fractionation column. FractionatedcDNA may then be ligated to pSPORT vector and subsequently transformedinto Escherichia coli DH5α. A library may then be plated, transferred tofilters, and amplified.

Screening of the cDNA library may be accomplished by hybridization withlabeled probes specific for the heavy and light chains. For example[32-P]-labeled probes such as MUCH-1 (5′-AGA CTG CAG GAG AGC TGG GAA GGTGTG CAC-3′) for heavy chain and MUCK-1 (5′-GAA GCA CAC GAC TGA GGC ACCTCC AGA TGT-3′) for light chain. Clones that are positive on a firstscreening may be transferred to duplicate plates and screened a secondtime with the same probes.

RNA isolation, cDNA synthesis, and amplification can be carried out asfollows. Total cell RNA can be prepared from a Mu-9 hybridoma cell line,using a total of about 10⁷ cells, according to Sambrook et al.,(Molecular Cloning: A Laboratory Manual, Second ed., Cold Spring HarborPress, 1989), which is incorporated by reference. First strand cDNA canbe reverse transcribed from total RNA conventionally, such as by usingthe SuperScript preamplification system (Gibco/BRL, Gaithersburg, Md.).Briefly, in a reaction volume of 20 μl, 50 ng of random hexamer primerscan be annealed to 5 μg of RNAs in the presence of 2 μl of 10× synthesisbuffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl, 25 mM MgCl₂, 1 mg/ml BSA],1 μl of 10 mM dNTP mix, 2 μl of 0.1 M DTT, and 200 units of SuperScriptreverse transcriptase. The elongation step is initially allowed toproceed at room temperature for 10 min followed by incubation at 42° C.for 50 min. The reaction can be terminated by heating the reactionmixture at 90° C. for 5 min.

Synthesizing and labeling the screening probes can be accomplished bywell-known means. Depending on the detection systems utilized, probelabeling will vary. Many kits for this purpose are commerciallyavailable. One method for 32-P labeling of oligonucleotides includes theuse of with [γ-³²P]ATP (Amersham Arlington Heights, Ill.) and T4polynucleotide kinase (New England Biolabs, Beverly, Mass.), followed bycolumn purification.

Preparation of a Chimeric Anti-CSAp Antibody

In general, to prepare chimeric anti-CSAp MAb, V_(H) and V κchains of aCSAp antibody may be obtained by methods such as those described aboveand amplified by PCR. In a preferred embodiment, the chimeric anti-CSApantibody is a Mu-9 antibody. The Vκ PCR products may be subcloned into apBR327 based staging vector (VKpBR) as described by leung et al.,Hybridoma, 13:469 (1994). The V_(H) PCR products may be subcloned into asimilar pBluescript-based staging vector (VHpBS). The fragmentscontaining the Vκ and V_(H) sequences, along with the promoter andsignal peptide sequences, can be excised from the staging vectors usingHindIII and BamHI restriction endonucleases. The Vκ fragments (about 600bp) can be subcloned into a mammalian expression vector (for example,pKh) conventionally. pKh is a pSVhyg-based expression vector containingthe genomic sequence of the human kappa constant region, an Ig enhancer,a kappa enhancer and the hygromycin-resistant gene. Similarly, the about800 bp V_(H) fragments can be subcloned into pG1g, a pSVgpt-basedexpression vector carrying the genomic sequence of the human IgG1constant region, an Ig enhancer and the xanthine-guanine phosphoribosyltransferase (gpt) gene. The two plasmids may be co-transfected intomammalian cells, such as Sp2/0-Ag14 cells, by electroporation andselected for hygromycin resistance. Colonies surviving selection areexpanded, and supernatant fluids monitored for production of cMu-9 mAbby an ELISA method. A transfection efficiency of about 1−10×10⁶ cells isdesirable. An antibody expression level of between 0.10 and 2.5 μg/m¹can be expected with this system.

Alternately, the Vκ and V_(H) expression cassettes can be assembled inthe modified staging vectors, VKpBR2 and VHpBS2, excised as XbaI/BamHIand XhoI/BamHI fragments, respectively, and subcloned into a singleexpression vector, such as pdHL2, as described by Gilles et al. J.Immunol. Methods 125:191 (1989), Losman et al., Clin. Cancer Res. 5:3101(1999) and in Losman et al., Cancer, 80:2660 (1997) for the expressionin Sp2/0-Ag14 cells. Another vector that is useful in the presentinvention is the GS vector, as described in Barnes et al.,Cytotechnology 32:109-123 (2000), which is preferably expressed in theNS0 cell line and CHO cells. Other appropriate mammalian expressionsystems are described in Werner et al., Arzneim.-Forsch./Drug Res.48(II), Nr. 8, 870-880 (1998

The Vκ and V_(H) sequences can be amplified by PCR as described byOrlandi et al., (Proc. Natl. Acad. Sci., U.S.A., 86: 3833 (1989)) whichis incorporated by reference. Vk sequences may be amplified using theprimers CK3BH and Vκ5-3 (Leung et al., BioTechniques, 15: 286 (1993),which is incorporated by reference), while V_(H) sequences can beamplified using the primer CH1B which anneals to the CH1 region ofmurine IgG, and VHIBACK (Orlandi et al., 1989 above). The PCR reactionmixtures containing 10 μl of the first strand cDNA product, 9 μl of10×PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCk₂, and0.01% (w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), can besubjected to 30 cycles of PCR. Each PCR cycle preferably consists ofdenaturation at 94° C. for 1 min, annealing at 50° C. for 1.5 min, andpolymerization at 72° C. for 1.5 min. Amplified Vk and VH fragments canbe purified on 2% agarose (BioRad, Richmond, Calif.).

Preparation of a Humanized Anti-CSAp Antibody

In a preferred embodiment, the humanized anti-CSAp antibody is ahumanized Mu-9 antibody. Once the sequences for the hMu-9Vκ and V_(H)domains are designed, CDR engrafting can be accomplished by genesynthesis using long synthetic DNA oligonucleotides as templates andshort oligonucleotides as primers in a PCR reaction. In most cases, theDNA encoding the Vκ or V_(H) domain will be approximately 350 bp long.By taking advantage of codon degeneracy, a unique restriction site mayeasily be introduced, without changing the encoded amino acids, atregions close to the middle of the V gene DNA sequence. For example, atDNA nucleotide positions 132-137 (amino acid positions 44-46) for thehMu-9 V_(H) domain, a unique XbaI site can be introduced whilemaintaining the originally designed amino acid sequence (see thesequence in FIG. 4A). Two long non-overlapping single-stranded DNAoligonucleotides (˜150 bp) upstream and downstream of the XbaI site canbe generated by automated DNA oligonucleotide synthesizer (Cyclone PlusDNA Synthesizer, Milligen-Biosearch). As the yields of full length DNAoligonucleotides may be expected to be low, they can be amplified by twopairs of flanking oligonucleotides in a PCR reaction. The primers can bedesigned with the necessary restriction sites to facilitate subsequentsequence assembly and subcloning. Primers for the oligonucleotidesshould contain overlapping sequence at the XbaI site so that theresultant PCR products can be joined in-frame at the XbaI site to form afull length DNA sequence encoding the hMu-9 VH domain. The ligation ofthe PCR products for the oligos at the XbaI site and their subcloninginto the PstII/BstEII sites of the staging vector, VHpBS, can becompleted in a single three-fragment ligation step. The subcloning ofthe correct sequence into VHpBS can be first analyzed by restrictiondigestion analysis and subsequently conformed by sequencing reactionaccording to Sanger et al., Proc. Natl. Acad. Sci. USA 74 5463 (1977).

The HindIII/BamHI fragment containing the Ig promoter, leader sequenceand the hMu-9 V_(H) sequence can be excised from the staging vector andsubcloned to the corresponding sites in a pSVgpt-based vector, pG1g,which contains the genomic sequence of the human IgG constant region, anIg enhancer and a gpt selection marker, forming the final expressionvector, hMu-9pG1g. Similar strategies can be employed for theconstruction of the hMu-9 VKc sequence. The restriction site chosen forthe ligation of the PCR products for the long oligonucleotides can beNru in this case.

The DNA sequence containing the Ig promoter, leader sequence and thehMu-9 VK sequence can be excised from the staging vector VKpBR bytreatment with BamHI/HindIII, and can be subcloned into thecorresponding sites of a pSVhyg-based vector, pKh, which contains thegenomic sequence of human kappa chain constant regions, a hygromycinselection marker, an Ig and a kappa enhancer, forming the finalexpression vector, hMu-9pKh.

The two plasmids can be co-transfected into an appropriate cell, e.g.,myeloma Sp2/0-Ag14, colonies selected for hygromycin resistance, andsupernatant fluids monitored for production of hMu-9 antibodies by, forexample, an ELISA assay, as described below. Alternately, the Vκ andV_(H) expression cassettes can be assembled in the modified stagingvectors, VKpBR2 and VHpBS2, excised as XbaI/BamHI and XhoI/BamHIfragments, respectively, and subcloned into a single expression vector,such as pdHL2, as described by Gilles et al., J. Immunol. Methods125:191 (1989), Losman et al., Clin. Cancer Res. 5:3101 (1999) and inLosman et al., Cancer, 80:2660 (1997) for the expression in Sp2/0-Ag14cells. Another vector that is useful in the present invention is the GSvector, as described in Barnes et al., Cytotechnology 32:109-123 (2000),which is preferably expressed in the NS0 cell line and CHO cells. Otherappropriate mammalian expression systems are described in Werner et al.,Arzneim.-Forsch./Drug Res. 48(II), Nr. 8, 870-880 (1998).

Transfection, and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of hMu-9pKh (light chain expressionvector) and 20 μg of hMu-9pG1g (heavy chain expression vector) can beused for the transfection of 5×10⁶ SP2/0 myeloma cells byelectroporation (BioRad, Richmond, Calif.) according to Co et al., J.Immunol., 148: 1149 (1992) which is incorporated by reference. Followingtransfection, cells may be grown in 96-well microtiter plates incomplete HSFM medium (GIBCO, Gaithersburg, Md.) at 37° C., 5% CO₂. Theselection process can be initiated after two days by the addition ofhygromycin selection medium (Calbiochem, San Diego, Calif.) at a finalconcentration of 500 μg/ml of hygromycin. Colonies typically emerge 2-3weeks post-electroporation. The cultures can then be expanded forfurther analysis.

Screening the Clones and Isolating Antibodies

Transfectoma clones that are positive for the secretion of chimeric orhumanized heavy chain can be identified by ELISA assay. Briefly,supernatant samples (100 μl) from transfectoma cultures are added intriplicate to ELISA microtiter plates precoated with goat anti-human(GAH)-IgG, F(ab′)₂ fragment-specific antibody (Jackson ImmunoResearch,West Grove, Pa.). Plates are incubated for 1 h at room temperature.Unbound proteins are removed by washing three times with wash buffer(PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)conjugated GAH-IgG, Fc fragment-specific antibodies (JacksonImmunoResearch, West Grove, Pa.) are added to the wells, (100 μl ofantibody stock diluted×10⁴, supplemented with the unconjugated antibodyto a final concentration of 1.0 μg/ml). Following an incubation of 1 h,the plates are washed, typically three times. A reaction solution, [100μl, containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4 N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 micron membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1 M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbancies at 280/260 nm. Peak fractions arepooled, dialyzed against PBS, and the antibody concentrated, forexample, with the Centricon 30 (Amicon, Beverly, Mass.). The antibodyconcentration is determined by ELISA, as before, and its concentrationadjusted to about 1 mg/ml using PBS. Sodium azide, 0.01% (w/v), isconveniently added to the sample as preservative.

The affinity of a chimeric, humanized or human anti-CSAp antibody may beevaluated using a direct binding assay or a competitive binding assay,as exemplified below.

Modifying/Optimizing the Recombinant Antibodies

As humanization sometimes results in a reduction or even loss ofantibody affinity, additional modification might be required in order torestore the original affinity (See, for example, Tempest et al.,Bio/Technology 9: 266 (1991); Verhoeyen et al., Science 239: 1534(1988)), which are incorporated by reference. Knowing that cMu-9exhibits a binding affinity comparable to that of its murinecounterpart, defective designs, if any, in the original version of hMu-9can be identified by mixing and matching the light and heavy chains ofcMu-9 to those of the humanized version. Preferably, some human residuesin the framework regions are replaced by their murine counterparts. Alsopreferred, a combination of framework sequences from 2 different humanantibodies, such as EU and NEWM are used for V_(H). For example, FR1-3can come from EU and FR 4 from NEWM.

Other modifications, such as Asn-linked glycosylation sites, can beintroduced into a chimerized, human, or humanized Mu-9 antibody byconventional oligonucleotide directed site-specific mutagenesis.Detailed protocols for oligonucleotide-directed mutagenesis and relatedtechniques for mutagenesis of cloned DNA are well known. For example,see Sambrook et al. and Ausubel et al. above.

For example, to introduce an Asn in position 18 of hMu-9 Vκ (FIG. 4B),one may alter codon 18 from CGA for Arg to AAC. To accomplish this, asingle stranded DNA template containing the antibody light chainsequence is prepared from a suitable strain of E. coli (e.g., dut, ung)in order to obtain a single strand DNA molecule containing a smallnumber of uracils in place of thymidine. Such a DNA template can beobtained by M13 cloning or by in vitro transcription using a SP6promoter. See, for example, Ausubel et al., eds., CURRENT PROTOCOLS 1NMOLECULAR BIOLOGY, John Wiley & Sons, NY, 1987. An oligonucleotidecontaining the mutated sequence is synthesized conventionally, annealedto the single-stranded template and the product treated with T4 DNApolymerase and T4 DNA ligase to produce a double-stranded DNA molecule.Transformation of wild type E. (dut⁺, ung⁺) cells with thedouble-stranded DNA provides an efficient recovery of mutated DNA.

Alternatively, an Asn-linked glycosylation site can be introduced intoan antibody light chain using an oligonucleotide containing the desiredmutation as the primer and DNA clones of the variable regions for the Vkchain, or by using RNA from cells that produce the antibody of interestas a template. Also see, Huse, in ANTIBODY ENGINEERING: A PRACTICALGUIDE, Boerrebaeck, ed., W. H. Freeman & Co., pp. 103-120, 1992.Site-directed mutagenesis can be performed, for example, using theTRANSFORMER™ kit (Clonetech, Palo Alto, Calif.) according to themanufacturer's instructions.

Alternatively, a glycosylation site can be introduced by synthesizing anantibody chain with mutually priming oligonucleotides, one suchcontaining the desired mutation. See, for example, Uhlmann, Gene 71: 29(1988); Wosnick et al., Gene 60: 115 (1988); Ausubel et al., above,which are incorporated by reference.

Although the general description above referred to the introduction ofan Asn glycosylation site in position 18 of the light chain of anantibody, it will occur to the skilled artisan that it is possible tointroduce Asn-linked glycosylation sites elsewhere in the light chain,or even in the heavy chain variable region.

Determining Antibody Binding Affinity

Comparative binding affinities of the isolated murine, human, humanizedand chimeric Mu-9 antibodies thus isolated may be determined by directradioimmunoassay. Mu-9 can be labeled with ¹³¹I or ¹²⁵I using thechloramine T method (see, for example, Greenwood et al., Biochem. J.,89: 123 (1963) which is incorporated by reference). The specificactivity of the iodinated antibody is typically adjusted to about 10μCi/μg. Unlabeled and labeled antibodies are diluted to the appropriateconcentrations using reaction medium (HSFM supplemented with 1% horseserum and 100 μg/ml gentamicin). The appropriate concentrations of bothlabeled and unlabeled antibodies are added together to the reactiontubes in a total volume of 100 μl. A culture of GW-39 tumor cells issampled and the cell concentration determined. The culture iscentrifuged and the collected cells washed once in reaction mediumfollowed by resuspension in reaction medium to a final concentration ofabout 10⁷ cells/ml. All procedures are carried out in the cold at 4° C.The cell suspension, 100 μl, is added to the reaction tubes. Thereaction is carried out at 4° C. for 2 h with periodic gentle shaking ofthe reaction tubes to resuspend the cells. Following the reactionperiod, 5 ml of wash buffer (PBS containing 1% BSA) is added to eachtube. The suspension is centrifuged and the cell pellet washed a secondtime with another 5 ml of wash buffer. Following centrifugation, theamount of remaining radioactivity remaining in the cell pellet isdetermined in a gamma counter (Minaxi, Packard Instruments, Sterling,Va.).

III. Production of Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab′)2, Fab′, Fab, Fv, sFv and the like. Otherantibody fragments include, but are not limited to: the F(ab)′₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab′ fragments, which can be generated by reducingdisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab′expression expression libraries can be constructed (Huse et al., 1989,Science, 246:1274-1281) to allow rapid and easy identification ofmonoclonal Fab′ fragments with the desired specificity. The presentinvention encompasses antibodies and antibody fragments.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,“Single Chain Antibody Variable Regions,” TIBTECH, Vol 9: 132-137(1991). These references are incorporated herein by reference.

To obtain high-affinity scFv, an scFv library with a large repertoirecan be constructed by isolating V-genes from non-immunized human donorsusing PCR primers corresponding to all known V_(H), V_(κ) and V_(λ) genefamilies. See, e.g., Vaughn et al., Nat. Biotechnol., 14: 309-314(1996). Following amplification, the V_(κ) and V_(λ) pools are combinedto form one pool. These fragments are ligated into a phagemid vector.The scFv linker, (Gly-Gly-Gly-Gly-Ser)₃, is then ligated into thephagemid upstream of the V_(L) fragment. The V_(H) and linker-V_(L)fragments are amplified and assembled on the J_(H) region. The resultingV_(H)-linker-V_(L) fragments are ligated into a phagemid vector. Thephagemid library can be panned using filters, as described above, orusing immunotubes (Nunc; Maxisorp). Similar results can be achieved byconstructing a combinatorial immunoglobulin library from lymphocytes orspleen cells of immunized rabbits and by expressing the scFv constructsin P. pastoris. See, e.g., Ridder et al., Biotechnology, 13: 255-260(1995). Additionally, following isolation of an appropriate scFv,antibody fragments with higher binding affinities and slowerdissociation rates can be obtained through affinity maturation processessuch as CDR3 mutagenesis and chain shuffling. See, e.g., Jackson et al.,Br. J. Cancer, 78: 181-188 (1998); Osbourn et al., Immunotechnology, 2:181-196 (1996).

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fab′monovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73:119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Preparation of a Bispecific Antibody

The bsAbs can be prepared by techniques known in the art, for example,anti-CSAp Ab and an anti-peptide Ab are both separately digested withpepsin to their respective F(ab′)₂s abd reduced with cystein to Fab′monomeric units. The Fab′ of anti-CSAp antibody is reacted with thecross-linker bis(maleimido) hexane to produce Fab′-maleimide moieties.The anti-peptide Ab-Fab′-SH is purified and reacted with the anti-CSApFab′-maleimide to generate the Fab′×Fab′ bi-specific Ab. Alternatively,the anti-peptide Fab′-SH fragment may be coupled with the anti-CSApF(ab′)₂ to generate a F(ab′)₂×Fab′ construct, or with anti-CSAp IgG togenerate an IgG×Fab′ bi-specific construct. In one embodiment, theIgG×Fab′ construct can be prepared in a site-specific manner byattaching the antipeptide Fab′ thiol group to anti-CSAp IgG heavy-chaincarbohydrate which has been periodate-oxidized, and subsequentlyactivated by reaction with a commercially available hydrazide-maleimidecross-linker. The component Abs used can be chimerized or humanized byknown techniques. A chimeric antibody is a recombinant protein thatcontains the variable domains and complementary determining regionsderived from a rodent antibody, while the remainder of the antibodymolecule is derived from a human antibody. Humanized antibodies arerecombinant proteins in which murine complementarity determining regionsof a monoclonal antibody have been transferred from heavy and lightvariable chains of the murine immunoglobulin into a human variabledomain.

A variety of recombinant methods can be used to produce bi-specificantibodies and antibody fragments. For example, bi-specific antibodiesand antibody fragments can be produced in the milk of transgeniclivestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63: 141-147,1998; U.S. Pat. No. 5,827,690. Two DNA constructs are prepared whichcontain, respectively, DNA segments encoding paired immunoglobulin heavyand light chains. The fragments are cloned into expression vectors whichcontain a promoter sequence that is preferentially expressed in mammaryepithelial cells. Examples include, but are not limited to, promotersfrom rabbit, cow and sheep casein genes, the cow α-lactoglobulin gene,the sheep β-lactoglobulin gene and the mouse whey acid protein gene.Preferably, the inserted fragment is flanked on its 3′ side by cognategenomic sequences from a mammary-specific gene. This provides apolyadenylation site and transcript-stabilizing sequences. Theexpression cassettes are coinjected into the pronuclei of fertilized,mammalian eggs, which are then implanted into the uterus of a recipientfemale and allowed to gestate. After birth, the progeny are screened forthe presence of both transgenes by Southern analysis. In order for theantibody to be present, both heavy and light chain genes must beexpressed concurrently in the same cell. Milk from transgenic females isanalyzed for the presence and functionality of the antibody or antibodyfragment using standard immunological methods known in the art. Theantibody can be purified from the milk using standard methods known inthe art.

A chimeric Ab is constructed by ligating the cDNA fragment encoding themouse light variable and heavy variable domains to fragment encoding theC domains from a human antibody. Because the C domains do not contributeto antigen binding, the chimeric antibody will retain the same antigenspecificity as the original mouse Ab but will be closer to humanantibodies in sequence. Chimeric Abs still contain some mouse sequences,however, and may still be immunogenic. A humanized Ab contains onlythose mouse amino acids necessary to recognize the antigen. This productis constructed by building into a human antibody framework the aminoacids from mouse complementarity determining regions.

Other recent methods for producing bsAbs include engineered recombinantAbs which have additional cysteine residues so that they crosslink morestrongly than the more common immunoglobulin isotypes. See, e.g.,FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997. Another approachis to engineer recombinant fusion proteins linking two or more differentsingle-chain antibody or antibody fragment segments with the needed dualspecificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163,1997. A variety of bi-specific fusion proteins can be produced usingmolecular engineering. In one form, the bi-specific fusion protein ismonovalent, consisting of, for example, a scFv with a single bindingsite for one antigen and a Fab fragment with a single binding site for asecond antigen. In another form, the bi-specific fusion protein isdivalent, consisting of, for example, an IgG with two binding sites forone antigen and two scFv with two binding sites for a second antigen.

Functional bi-specific single-chain antibodies (bscAb), also calleddiabodies, can be produced in mammalian cells using recombinant methods.See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995. Forexample, bscAb are produced by joining two single-chain Fv fragments viaa glycine-serine linker using recombinant methods. The V light-chain(V_(L)) and V heavy-chain (V_(H)) domains of two antibodies of interestare isolated using standard PCR methods. The V_(L) and V_(H) cDNA'sobtained from each hybridoma are thenjoined to form a single-chainfragment in a two-step fusion PCR. The first PCR step introduces the(Gly₄-Ser₁)₃ linker, and the second step joins the V_(L) and V_(H)amplicons. Each single chain molecule is then cloned into a bacterialexpression vector. Following amplification, one of the single-chainmolecules is excised and sub-cloned into the other vector, containingthe second single-chain molecule of interest. The resulting bscAbfragment is subcloned into an eukaryotic expression vector. Functionalprotein expression can be obtained by transfecting the vector intochinese hamster ovary cells. Bi-specific fusion proteins are prepared ina similar manner. Bi-specific single-chain antibodies and bi-specificfusion proteins are included within the scope of the present invention.Diabody, triabody and tetrabody bispecific fusion proteins produced inE. coli and yeast are described in U.S. Provisional Application No.60/345,641, 60/328,835 and 60/342,103, and are incorporated herein byreference.

Bi-specific fusion proteins linking two or more different single-chainantibodies or antibody fragments are produced in similar manner.

Large quantities of bscAb and fusion proteins can be produced usingEscherichia coli expression systems. See, e.g., Zhenping et al.,Biotechnology, 14: 192-196, 1996. A functional bscAb can be produced bythe coexpression in E. coli of two “cross-over” scFv fragments in whichthe V_(L) and V_(H) domains for the two fragments are present ondifferent polypeptide chains. The V light-chain (V_(L)) and Vheavy-chain (V_(H)) domains of two antibodies of interest are isolatedusing standard PCR methods. The cDNA's are then ligated into a bacterialexpression vector such that C-terminus of the V_(L) domain of the firstantibody of interest is ligated via a linker to the N-terminus of theV_(H) domain of the second antibody. Similarly, the C-terminus of theV_(L) domain of the second antibody of interest is ligated via a linkerto the N-terminus of the V_(H) domain of the first antibody. Theresulting dicistronic operon is placed under transcriptional control ofa strong promoter, e.g., the E. coli alkaline phosphatase promoter whichis inducible by phosphate starvation. Alternatively, single-chain fusionconstructs have successfully been expressed in E. coli using the lacpromoter and a medium consisting of 2% glycine and 1% Triton X-100. See,e.g., Yang et al., Appl. Environ. Microbiol., 64: 2869-2874, 1998. An E.coli/, heat-stable, enterotoxin II signal sequence is used to direct thepeptides to the periplasmic space. After secretion, the two peptidechains associate to form a non-covalent heterodimer which possesses bothantigen binding specificities. The bscAb is purified using standardprocedures known in the art, e.g., Staphylococcal protein Achromatography.

Functional bscAb and fusion proteins also can be produced in the milk oftransgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63:141-147, 1998; U.S. Pat. No. 5,827,690. The bscAb fragment, obtained asdescribed above, is cloned into an expression vector containing apromoter sequence that is preferentially expressed in mammary epithelialcells. Examples include, but are not limited to, promoters from rabbit,cow and sheep casein genes, the cow α-lactoglobulin gene, the sheepβ-lactoglobulin gene and the mouse whey acid protein gene. Preferably,the inserted bscAb is flanked on its 3′ side by cognate genomicsequences from a mammary-specific gene. This provides a polyadenylationsite and transcript-stabilizing sequences. The expression cassette isthen injected into the pronuclei of fertilized, mammalian eggs, whichare then implanted into the uterus of a recipient female and allowed togestate. After birth, the progeny are screened for the presence of theintroduced DNA by Southern analysis. Milk from transgenic females isanalyzed for the presence and functionality of the bscAb using standardimmunological methods known in the art. The bscAb can be purified fromthe milk using standard methods known in the art. Transgenic productionof bscAb in milk provides an efficient method for obtaining largequantities of bscAb.

Functional bscAb and fusion proteins also can be produced in transgenicplants. See, e.g., Fiedler et al., Biotech., 13: 1090-1093, 1995;Fiedler et al., Immunotechnology, 3: 205-216, 1997. Such productionoffers several advantages including low cost, large scale output andstable, long term storage. The bscAb fragment, obtained as describedabove, is cloned into an expression vector containing a promotersequence and encoding a signal peptide sequence, to direct the proteinto the endoplasmic recticulum. A variety of promoters can be utilized,allowing the practitioner to direct the expression product to particularlocations within the plant. For example, ubiquitous expression intobacco plants can be achieved by using the strong cauliflower mosaicvirus 35S promoter, while organ specific expression is achieved via theseed specific legumin B4 promoter. The expression cassette istransformed according to standard methods known in the art.Transformation is verified by Southern analysis. Transgenic plants areanalyzed for the presence and functionality of the bscAb using standardimmunological methods known in the art. The bscAb can be purified fromthe plant tissues using standard methods known in the art.

Additionally, transgenic plants facilitate long term storage of bscAband fusion proteins. Functionally active scFv proteins have beenextracted from tobacco leaves after a week of storage at roomtemperature. Similarly, transgenic tobacco seeds stored for 1 year atroom temperature show no loss of scFv protein or its antigen bindingactivity.

Functional bscAb and fusion proteins also can be produced in insectcells. See, e.g., Mahiouz et al., J. Immunol. Methods, 212: 149-160(1998). Insect-based expression systems provide a means of producinglarge quantities of homogenous and properly folded bscAb. Thebaculovirus is a widely used expression vector for insect cells and hasbeen successfully applied to recombinant antibody molecules. See, e.g.,Miller, L. K., Ann. Rev. Microbiol., 42: 177 (1988); Bei et al., J.Immunol. Methods, 186: 245 (1995). Alternatively, an inducibleexpression system can be utilized by generating a stable insect cellline containing the bscAb construct under the transcriptional control ofan inducible promoter. See, e.g., Mahiouz et al., J. Immunol. Methods,212: 149-160 (1998). The bscAb fragment, obtained as described above, iscloned into an expression vector containing the Drosphilametallothionein promoter and the human HLA-A2 leader sequence. Theconstruct is then transfected into D. melanogaster SC-2 cells.Expression is induced by exposing the cells to elevated amounts ofcopper, zinc or cadmium. The presence and functionality of the bscAb isdetermined using standard immunological methods known in the art.Purified bscAb is obtained using standard methods known in the art.

Preferred bispecific antibodies of the instant invention are those whichincorporate the Fv of MAb Mu-9 and the Fv of MAb 679 and their human,chimerized or humanized counterparts. Accordingly, an anti-CSAp antibodyfragments are also contemplated in the present invention. Preferably,the anti-CSAp antibody fragment is a Mu-9 antibody fragment. Alsopreferred are bispecific antibodies which incorporate one or more of theCDRs of Mu-9.

IV. Antibodies for Treatment and Diagnosis/Detection

Humanized, Chimeric and Human Anti-CSAp Antibodies for Treatment andDiagnosis/Detection.

Contemplated in the present invention is the use of humanized, chimericand human anti-CSAp antibodies and fragments thereof in therapeutic anddiagnostic/detection methods. Preferably, the chimeric, humanized andhuman anti-CSAp antibodies and fragments thereof are chimeric, humanizedor human Mu-9 antibodies. Still preferred, the chimeric, humanized andhuman Mu-9 antibodies and fragments thereof of the present invention areused in methods for treating malignancies. For example, a malignancy ofparticular interest in this patent is a cancer of the gastrointestinalsystem, more preferably of the colon and rectum, pancreas, as well asovarian cancer.

The compositions for treatment contain at least one naked humanized,chimeric or human anti-CSAp antibody or fragment thereof alone or incombination with other anti-CSAp antibodies or antibody fragmentsthereof, such as other anti-CSAp humanized, chimeric or humanantibodies. The present invention also contemplates treatment with atleast one naked humanized, chimeric or human anti-CSAp antibody orfragment thereof in combination with other antibodies or antibodyfragments thereof that are not anti-CSAp antibodies, whereby these otherantibodies can be administered unconjugated (naked) or conjugated with atherapeutic compound. For example, other antibodies suitable forcombination therapy include, but are not limited to,carcinoma-associated antibodies and fragments thereof such as antibodiesagainst CEA, EGP-1, Ga 733 antigen target, such as for antibodies EGP-2,17-1A, KS14 and Ep-CAM, MUC1, MUC2, MUC 3, MUC-4, PAM-4, KC4, TAG-72,EGFR, HER2/neu, BrE3, Le-Y, KS-1, A3, anti-necrosis antibodies, and theA33 antibody determinant. Suitable antibodies could also include thosetargeted against oncogene markers or products, or antibodies againsttumor-vasculature markers, such as the angiogenesis factor, VEGF, andantibodies against certain immune response modulators, such asantibodies to CD40. Additionally, treatment can be effected with atleast one humanized, chimeric or human anti-CSAp immunoconjugate orfragment thereof alone or in combination with other anti-CSAp antibodiesor antibody fragments thereof, such as other anti-CSAp humanized,chimeric or human antibodies. Still preferred, compositions fortreatment can contain at least one humanized, chimeric or humananti-CSAp immunoconjugate or fragment thereof in combination with otherantibodies or antibody fragments thereof that are not anti-CSApantibodies, these being either naked or conjugated to a therapeuticagent.

Similarly, conjugated and naked anti-CSAp humanized, chimeric or humanantibodies may be used alone or may be administered with, butunconjugated to, the various diagnostic or therapeutic agents describedherein. Also, naked or conjugated antibodies to the same or differentepitope or antigen may be also combined with one or more of theantibodies of the present invention.

Accordingly, the present invention contemplates the administration ofhumanized, chimeric and human Mu-9 antibodies and fragments thereofalone, as a naked antibody, or administered as a multimodal therapy.Multimodal therapies of the present invention further includeimmunotherapy with naked or conjugated CSAp antibodies supplemented withadministration of other antibodies in the form of naked antibodies,fusion proteins, or as immunoconjugates. For example, a humanized,chimeric or human Mu-9 antibody may be combined with another nakedhumanized, naked chimeric or naked human Mu-9 antibody, or a humanized,chimeric or human Mu-9 antibody immunoconjugate, such as a humanized,chimeric or human Mu-9 antibody conjugated to an isotope, one or morechemotherapeutic agents, cytokines, enzymes, enzyme-inhibitors, hormonesor hormone antagonists, metals, toxins, or a combination thereof. Afusion protein of a humanized, chimeric or human Mu-9 antibody and atoxin or may also be used in this invention. Many different antibodycombinations may be constructed, either as naked antibodies or as partlynaked and partly conjugated with a therapeutic agent or immunomodulator,or merely in combination with another therapeutic agents, such as acytotoxic drug or with radiation.

The compositions for treatment contain at least one humanized, chimericor human monoclonal CSAp antibody or fragment thereof alone or incombination with other antibodies and fragments thereof, such as othernaked or conjugated humanized, chimeric, or human antibodies, fusionproteins, or therapeutic agents. In particular, combination therapy witha fully human antibody is also contemplated and is produced by themethods as set forth above.

Naked or conjugated antibodies and fragments thereof to the same ordifferent epitope or antigen may be also combined with one or more ofthe antibodies or fragments thereof of the present invention. Forexample, a humanized, chimeric or human naked Mu-9 antibody may becombined with another naked humanized, naked chimeric or naked humanMu-9 antibody, a humanized; chimeric or human naked Mu-9 antibody may becombined with a Mu-9 immunoconjugate, a naked Mu-9 antibody may becombined with a different antibody radioconjugate or an different nakedantibody may be combined with a humanized, chimeric or human Mu-9antibody conjugated to an isotope, or to one or more chemotherapeuticagents, cytokines, toxins, enzymes, enzyme inhibitors, hormones, hormoneantagonists, or a combination thereof A fusion protein of a humanized,chimeric or human Mu-9 antibody and a toxin or immunomodulator may alsobe used in this invention. Many different antibody combinations,targeting at least two different antigens may be constructed, either asnaked antibodies or as partly naked and partly conjugated with atherapeutic agent or immunomodulator, or merely in combination withanother therapeutic agents, such as a cytotoxic drug or with radiation.

Multimodal therapies of the present invention further includeimmunotherapy with naked Mu-9 antibodies or fragments thereofsupplemented with administration of antibodies against antigensexpressed by colorectal or ovarian carcinomas in the form of nakedantibodies, fusion proteins, immunoconjugates, and fragments thereof. Ina preferred embodiment, antibodies or fragments thereof for multimodaltherapy include, but are not limited to, antibodies against CEA, EGP-1,EGP-2, TAG-72, MUC1, MUC2, MUC3, MUC4, KC4, PAM4, EGFR, BrE3, Le-Y,KS-1, A3, the A33 antibody and HER2/neu antibodies and fragmentsthereof, as well as antibodies against angiogenesis factors (e.g., VEGF)or antibodies against oncogene markers or products, as well asantibodies against immunomodulators, (e.g., CD40). These antibodiesinclude polyclonal, monoclonal, chimeric, human or humanized antibodiesand fragments thereof that recognize at least one epitope on theseantigenic determinants.

In another form of multimodal therapy, subjects receive naked antibodiesor fragments thereof, and/or immunoconjugates or fragments thereof, inconjunction with standard cancer chemotherapy. 5-fluorouracil incombination with folinic acid, alone or in combination with irinotecan(CPT-11), is a regimen used to treat colorectal cancer. Other suitablecombination chemotherapeutic regimens are well known, such as withoxaliplatin alone, or in combination with these other drugs, to those ofskill in the art. In ovarian cancer, still other chemotherapeutic agentsmay be preferred, such as any one of the taxanes and platinum agents,Thio-TEPA and other alkylating agents (e.g., chlorambucil), as well asgemcitabine and other more recent classes of cytotoxic drugs. In apreferred multimodal therapy, both chemotherapeutic drugs and cytokinesare co-administered with an antibody, immunoconjugate or fusion proteinaccording to the present invention. The cytokines, chemotherapeuticdrugs and antibody or immunoconjugate can be administered in any order,or together.

A variety of other chemotherapeutic agents may be used in forcombination treatment or for making immunoconjugates. Suchchemotherapeutic agents include, but are not limited to, adriamycin,dactinomycin, mitomycin, caminomycin, daunomycin, doxorubicin,tamoxifen, taxol and other taxanes, taxotere, vincristine, vinblastine,vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU), cytosinearabinoside, cyclophohphamide, thiotepa, methotrexate, camptothecin,actinomycin-D, mitomycin C, cisplatin (CDDP), aminopterin,combretastatin(s), neomycin, podophyllotoxin(s), TNF-α, α₂β₃antagonists, calcium ionophores, calcium-flux inducing agents andderivatives and prodrugs thereof. Anti-metabolites such as cytosinearabinoside, amethopterin; anthracyclines; vinca alkaloids and otheralkaloids; antibiotics, demecolcine; etopside; mithramycin; and otheranti-tumor alkylating agents are also contemplated for use with theantibodies of the present invention.

In addition, a therapeutic composition of the present invention cancontain a mixture or hybrid molecules of monoclonal naked Mu-9antibodies or their fragments directed to different, non-blockingepitopes on the CSAp molecule. Accordingly, the present inventioncontemplates therapeutic compositions comprising a mixture of monoclonalMu-9 antibodies or their fragments that bind at least two CSAp epitopes.Additionally, the therapeutic composition described herein may contain amixture of Mu-9 antibodies and fragments thereof with varying CDRsequences.

Naked Antibody Therapy

A therapeutically effective amount of the naked chimeric, humanized orhuman anti-CSAp antibodies, or their fragments can be formulated in apharmaceutically acceptable excipient. The efficacy of the naked Mu-9antibodies can also be enhanced by supplementing naked antibodies withone or more other naked antibodies (such as against tumor-associatedantigens, or also agonist or antagonist antibodies to immunomodulators,such as CD40 antigen or ligand), with one or more immunoconjugates ofMu-9, with one or more immunoconjugates of the antibodies againsttumor-associated antigens other than CSAp, and conjugated withtherapeutic agents, including drugs, toxins, cytokines,immunomodulators, hormones, hormone antagonists, enzymes, enzymeinhibitors, therapeutic radionuclides, etc., with one or moretherapeutic agents, including drugs, toxins, cytokines,immunomodulators, hormones, enzymes, enzyme inhibitors, therapeuticradionuclides, etc., administered concurrently or sequentially oraccording to a prescribed dosing regimen, with the Mu-9 antibodies.

Humanized, Chimeric and Human Anti-CSAp Immunoconjugates

Alternatively, conjugates of the Mu-9 antibodies or fragments thereof ofthe present invention may be administered. For therapy, these conjugatespreferably contain a cytotoxic agent. More preferably, the cytotoxicagent is a toxin. An immunoconjugate, as described herein, is a moleculecomprising an antibody component and a therapeutic or diagnostic agent,including a peptide which may bear the diagnostic or therapeutic agent.An immunoconjugate retains the immunoreactivity of the antibodycomponent, i.e., the antibody moiety has about the same or slightlyreduced ability to bind the cognate antigen after conjugation as beforeconjugation.

A wide variety of diagnostic/detection and therapeutic reagents can beadvantageously conjugated to the antibodies and fragments thereof, ofthe invention. The therapeutic agents recited here are those agents thatalso are useful for administration separately with the naked antibody asdescribed above. Therapeutic agents include, for example,chemotherapeutic drugs such as vinca alkaloids and other alkaloids,anthracyclines, epidophyllotoxins, taxanes, antimetabolites, alkylatingagents, antibiotics, COX-2 inhibitors, antimitotics, antiangiogenic andapoptotoic agents, particularly doxorubicin, methotrexate, taxol,CPT-11, camptothecans, and others from these and other classes ofanticancer agents, and the like. Other useful cancer chemotherapeuticdrugs for the preparation of immunoconjugates and antibody fusionproteins include nitrogen mustards, alkyl sulfonates, nitrosoureas,triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine analogs,purine analogs, platinum coordination complexes, hormones, toxins (e.g.,RNAse, Psudomonas exotoxin), and the like. Suitable chemotherapeuticagents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.(Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co.1985), as well as revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated. For example, a therapeutically useful immunoconjugate can beobtained by conjugating a photoactive agent or dye to an antibodycomposite. Fluorescent compositions, such as fluorochrome, and otherchromogens, or dyes, such as porphyrins sensitive to visible light, havebeen used to detect and to treat lesions by directing the suitable lightto the lesion. In therapy, this has been tenned photoradiation,phototherapy, or photodynamic therapy (Jori et al. (eds.), PHOTODYNAMICTHERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van denBergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodieshave been coupled with photoactivated dyes for achieving phototherapy.Mew et al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380(1985); Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744 (1986);idem., Photochem. Photobiol. 46:83 (1987); Hasan et al., Prog. Clin.Biol. Res. 288:471 (1989); Tatsuta et al, Lasers Surg. Med. 9:422(1989); Pelegrin et al., Cancer 67:2529 (1991). However, these earlierstudies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes.

A toxin, such as Pseudomonas exotoxin, may also be complexed to or formthe therapeutic agent portion of an immunoconjugate of the Mu-9 antibodyof the present invention, or when used in combination with the nakedMu-9 antibody or conjugates of the Mu-9 antibody, also complexed to theother, non-CSAp antibodies used in this invention. Other toxins suitablyemployed in the preparation of such conjugates or other fusion proteins,include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al, Cell 47:641 (1986), and Goldenberg, C A—A Cancer Journalfor Clinicians 44:43 (1994). Additional toxins suitable for use in thepresent invention are known to those of skill in the art and aredisclosed in U.S. Pat. No. 6,077,499, which is incorporated in itsentirety by reference. These can be derived, for example, from animal,plant and microbial sources, or chemically or recombinantly engineered.The toxin can be a plant, microbial, or animal toxin, or a syntheticvariation thereof.

An immunomodulator, such as a cytokine may also be conjugated to, orform the therapeutic agent portion of the anti-CSAp immunoconjugate, orbe administered unconjugated to the chimeric, humanized or humananti-CSAp antibodies of the present invention. As used herein, the term“immunomodulator” includes cytokines, stem cell growth factors,lymphotoxins, such as tumor necrosis factor (TNF), and hematopoieticfactors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3,IL-6, IL-10, IL-12 and IL-18), colony stimulating factors (e.g.,granulocyte-colony stimulating factor (G-CSF) and granulocytemacrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,interferons-α, -β and -γ), the stem cell growth factor designated “S1factor,” erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18,interferon-γ, TNF-α, and the like. Alternatively, subjects can receivenaked Mu-9 antibodies and a separately administered cytokine, which canbe administered before, concurrently or after administration of thenaked Mu-9 antibodies. The Mu-9 antibody may also be conjugated to theimmunomodulator. The immunomodulator may also be conjugated to a hybridantibody consisting of one or more antibodies binding to differentantigens. Such an antigen may also be an immunomodulator. For example,CD40 or other immunomodulators may be administered in combination withthe anti-CSAp or anti-CSAp/non-CSAp antibody combination eithertogether, before or after the antibody combinations are administered.The Mu-9 antibody may also be used in combination with, or conjugatedto, as a fusion protein, an immunomodulating antibody, such as againstCD40.

Furthermore, the present invention includes methods of diagnosing ordetecting a malignancy in a subject. Diagnosis/detection may beaccomplished by administering a diagnostically effective amount of adiagnostic/detection conjugate, formulated in a pharmaceuticallyacceptable excipient, and detecting said label. For example, radioactiveand non-radioactive agents can be used as diagnostic agents. A suitablenon-radioactive diagnostic agent is a contrast agent suitable formagnetic resonance imaging, a radiopaque compound for X-rays or computedtomography, or a contrast agent suitable for ultrasound. Magneticimaging agents include, for example, non-radioactive metals, such asmanganese, iron and gadolinium, complexed with metal-chelatecombinations that include 2-benzyl-DTPA and its monomethyl andcyclohexyl analogs, when used along with the antibodies of theinvention. See U.S. Ser. No. 09/921,290 filed on Oct. 10, 2001, which isincorporated in its entirety by reference. In a preferred embodiment,the contrast agent is an ultrasound-enhancing agent. Still preferred,the ultrasound-enhancing agent is a liposome. Radiopaque and contrastmaterials are used for enhancing X-rays and computed tomography, andinclude iodine compounds, barium compounds, gallium compounds, thalliumcompounds, etc. Specific compounds include barium, diatrizoate,ethiodized oil, gallium citrate, iocarnic acid, iocetamic acid,iodamide, iodipamide, iodoxamic acid, iogulamide, iohexyl, iopamidol,iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamidemeglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid,iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine,metrizamide, metrizoate, propyliodone, and thallous chloride.

Furthermore, a radiolabeled antibody or immunoconjugate may comprise aγ-emitting radioisotope or a positron-emitter useful for diagnosticimaging. Examples of diagnostic/detection agents include diverse labels,radionuclides, chelators, dyes, contrast agents, fluorescent compounds,chromagens, and other marker moieties. Radionuclides useful for positronemission tomography include, but are not limited to: F-18, Mn-51,Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68, As-72, Br-75, Br-76, Rb-82m,Sr-83, Y-86, Zr-89, Tc-94m, In-110, I-120, and I-124. Total decayenergies of useful positron-emitting radionuclides are preferably <2,000keV, more preferably under 1,000 keV, and most preferably <700 keV.Radionuclides useful as diagnostic agents utilizing gamma-ray detectioninclude, but are not limited to: Cr-51, Co-57, Co-58, Fe-59, Cu-67,Ga-67, Se-75, Ru-97, Tc-99m, In-111, In-114m, I-123, I-125, I-131,Yb-169, Hg-197, and Tl-201. Decay energies of useful gamma-ray emittingradionuclides are preferably 20-2000 keV, more preferably 60-600 keV,and most preferably 100-300 keV.

Additionally, radionuclides suitable for treating a diseased tissueinclude, but are not limited to, P-32, P-33, Sc-47, Fe-59, Cu-64, Cu-67,Se-75, As-77, Sr-89, Y-90, Mo-99, R^(h)-105, Pd-109, Ag-111, I-125,I-131, Pr-142, Pr-143, Pm-149, Sm-153, Th-161, Ho-166, Er-169, Lu-177,Re-186, Re-188, Re-189, Ir-194, Au-198, Au-199, Pb-211, Pb-212, andBi-213, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119,I-125, Ho-161, Os-189m, Ir-192, Dy-152, At-211, Bi-212, Ra-223, Rn-219,Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255.

Suitable diagnostic imaging isotopes are usually in the range of 20 to2,000 keV, while suitable therapeutic radionuclides are usually in therange of 20 to 10,000 keV. See for example, U.S. patent applicationentitled “Labeling Targeting Agents with Gallium-68”—Inventors G. L.Griffiths and W. J. McBride, (U.S. Provisional Application No.60/342,104), which discloses positron emitters, such as ¹⁸F, ⁶⁸Ga,^(94m)Tc. and the like, for imaging purposes and which is incorporatedin its entirety by reference.

Bispecific Antibody Therapy

The present invention also encompasses the use of the bsAb and atherapeutic agent associated with the linker moieties discussed above inintraoperative, intravascular, and endoscopic tumor and lesiondetection, biopsy and therapy as described in U.S. Pat. No. 6,096,289,and incorporated herein by reference.

The antibodies and antibody fragments of the present invention can beemployed not only for therapeutic or imaging purposes, but also as aidsin performing research in vitro. For example, the bsAbs of the presentinvention can be used in vitro to ascertain if a targetable constructcan form a stable complex with one or more bsAbs. Such an assay wouldaid the skilled artisan in identifying targetable constructs which formstable complexes with bsAbs. This would, in turn, allow the skilledartisan to identify targetable constructs which are likely to besuperior as therapeutic and/or imaging agents.

The assay is advantageously performed by combining the targetableconstruct in question with at least two molar equivalents of a bsAb.Following incubation, the mixture is analyzed by size-exclusion HPLC todetermine whether or not the construct has bound to the bsAb.Alternatively, the assay is performed using standard combinatorialmethods wherein solutions of various bsAbs are deposited in a standard96-well plate. To each well, is added solutions of targetableconstruct(s). Following incubation and analysis, one can readilydetermine which construct(s) bind(s) best to which bsAb(s).

It should be understood that the order of addition of the bsAb to thetargetable construct is not crucial; that is, the bsAb may be added tothe construct and vice versa. Likewise, neither the bsAb nor theconstruct needs to be in solution; that is, they may be added either insolution or neat, whichever is most convenient. Lastly, the method ofanalysis for binding is not crucial as long as binding is established.Thus, one may analyze for binding using standard analytical methodsincluding, but not limited to, FABMS, high-field NMR or otherappropriate method in conjunction with, or in place of, size-exclusionHPLC.

The present invention provides a bispecific antibody or antibodyfragment having at least a binding region that specifically binds atargeted cell marker and at least one other binding region thatspecifically binds a targetable conjugate. The targetable conjugatecomprises a carrier portion which comprises or bears at least oneepitope recognized by at least one binding region of the bispecificantibody or antibody fragment.

For example, the anti-CSAp antibodies and fragments thereof, as well asother antibodies with different specificities and fragments thereof, foruse in combination therapy, described herein, can also be made asmultispecific antibodies (comprising at least one binding site to a CSApepitope or antigen and at least one binding site to another epitope onCSAp or another antigen) and multivalent antibodies (comprising mutliplebinding sites to the same epitope or antigen).

A variety of recombinant methods can be used to produce bispecificantibodies and antibody fragments as described above.

In a preferred embodiment, the multivalent antibody is a Mu-9 antibody.A Mu-9 multivalent antibody is also contemplated in the presentinvention. This multivalent antibody is constructed by association of afirst and a second polypeptide. The first polypeptide comprises a firstsingle chain Fv molecule covalently linked to a firstimmunoglobulin-like domain which preferably is an immunoglobulin lightchain variable region domain. The second polypeptide comprises a secondsingle chain Fv molecule covalently linked to a secondimmunoglobulin-like domain which preferably is an immunoglobulin heavychain variable region domain. Each of the first and second single chainFv molecules forms a target binding site, and the first and secondimmunoglobulin-like domains associate to form a third target bindingsite.

A single chain Fv molecule with the VL-L-VH configuration, wherein L isa linker, may associate with another single chain Fv molecule with theVH-L-VL configuration to form a bivalent dimer. In this case, the VLdomain of the first scFv and the VH domain of the second scFv moleculeassociate to form one target binding site, while the VH domain of thefirst scFv and the VL domain of the second scFv associate to form theother target binding site.

Another embodiment of the present invention is a Mu-9 bispecific,trivalent antibody comprising two heterologous polypeptide chainsassociated non-covalently to form three binding sites, two of which haveaffinity for one target and a third which has affinity for a hapten thatcan be made and attached to a carrier for a diagnostic and/ortherapeutic agent. Preferably, the binding protein has two CSAp bindingsites and one other antigen binding site. The bispecific, trivalenttargeting agents have two different scFvs, one scFv contains two V_(H)domains from one antibody connected by a short linker to the V_(L)domain of another antibody and the second scFv contains two V_(L)domains from the first antibody connected by a short linker to the V_(H)domain of the other antibody. The methods for generating multivalent,multispecific agents from V_(H) and V_(L) domains provide thatindividual chains synthesized from a DNA plasmid in a host organism arecomposed entirely of V_(H) domains (the V_(H)-chain) or entirely ofV_(L) domains (the V_(L)-chain) in such a way that any agent ofmultivalency and multispecificity can be produced by non-covalentassociation of one V_(H)-chain with one V_(L)-chain. For example,forming a trivalent, trispecific agent, the V_(H)-chain will consist ofthe amino acid sequences of three V_(H) domains, each from an antibodyof different specificity, joined by peptide linkers of variable lengths,and the V_(L)-chain will consist of complementary V_(L) domains, joinedby peptide linkers similar to those used for the V_(H)-chain. Since theV_(H) and V_(L) domains of antibodies associate in an anti-parallelfashion, the preferred method in this invention has the V_(L) domains inthe V_(L)-chain arranged in the reverse order of the V_(H) domains inthe V_(H)-chain.

Bispecific antibodies and fragments thereof of the present invention areuseful in pretargeting methods and provide a preferred way to delivertwo therapeutic agents or two diagnostic/detection agents to a subject.U.S. Ser. No. 09/382,186 discloses a method of pretargeting using abispecific antibody, in which the bispecific antibody is labeled with¹²⁵I and delivered to a subject, followed by a divalent peptide labeledwith ^(99m)Tc. The delivery results in excellent tumor/normal tissueratios for ¹³¹I and ^(99m)Tc, thus showing the utility of two diagnosticradioisotopes. Any combination of known therapeutic agents or diagnosticagents can be used to label the antibodies and antibody fusion proteins.The binding specificity of the antibody component of the mAb conjugate,the efficacy of the therapeutic agent or diagnostic agent and theeffector activity of the Fc portion of the antibody can be determined bystandard testing of the conjugates.

Preparation of Humanized, Chimeric and Human Mu-9 Immunoconjugates

Any of the anti-CSAp antibodies or fragments thereof or antibody fusionproteins or fragments thereof of the present invention can be conjugatedwith one or more therapeutic or diagnostic agents. Generally, onetherapeutic or diagnostic agent is attached to each antibody or antibodyfragment but more than one therapeutic agent or diagnostic agent can beattached to the same antibody or antibody fragment. The antibody fusionproteins of the present invention comprise two or more antibodies orfragments thereof and each of the antibodies that compose this fusionprotein can contain a therapeutic agent or diagnostic agent. In otherwords, the antibody fusion protein or fragment thereof can comprise atleast one first anti-CSAp MAb or fragment thereof and at least onesecond MAb or fragment thereof that is not an anti-CSAp MAb. Preferably,the second MAb is a carcinoma-associated antibody, such as an antibodyagainst CEA, EGP-1, EGP-2, MUC1, MUC2, MUC3, MUC4, PAM-4, KC4, TAG-72,EGFR, HER2/neu, BrE3, Le-Y, A3, KS-1, VEGF and other angiogenesisantibodies, oncogene antibodies, anti-necrosis antibodies, or theantibody A33. Additionally, one or more of the antibodies of theantibody fusion protein can have more than one therapeutic ordiagnostic/detection agent attached. Further, the therapeutic agents donot need to be the same but can be different therapeutic agents; forexample, one can attach a drug and a radioisotope to the same fusionprotein. Particulary, an IgG can be radiolabeled with ¹³¹I and attachedto a drug. The ¹³¹I can be incorporated into the tyrosine of the IgG andthe drug attached to the epsilon amino group of the IgG lysines. Boththerapeutic and diagnostic agents also can be attached to reduced SHgroups and to the carbohydrate side chains.

Also preferred, the antibody fusion protein of the present inventioncomprises at least two anti-CSAp monoclonal antibodies or fragmentsthereof, and these may be to different epitopes of the CSAp antigen orof different human immunoglobulin backbone sequences (or IgGs).

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation. As analternative, such peptides can be attached to the antibody componentusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic agent can be conjugated via a carbohydrate moiety in the Fcregion of the antibody. The carbohydrate group can be used to increasethe loading of the same peptide that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different peptide.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

The Fc region is absent if the antibody used as the antibody componentof the immunoconjugate is an antibody fragment. However, it is possibleto introduce a carbohydrate moiety into the light chain variable regionof a full-length antibody or antibody fragment. See, for example, Leunget al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No.5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, all of whichare incorporated in their entirety by reference. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

V. Constructs Targetable to Antibodies

The targetable construct can be of diverse structure, but is selectednot only to avoid eliciting an immune responses, but also for rapid invivo clearance when used within the bsAb targeting method. Hydrophobicagents are best at eliciting strong immune responses, whereashydrophilic agents are preferred for rapid in vivo clearance; thus, abalance between hydrophobic and hydrophilic needs to be established.This is accomplished, in part, by relying on the use of hydrophilicchelating agents to offset the inherent hydrophobicity of many organicmoieties. Also, subunits of the targetable construct may be chosen whichhave opposite solution properties, for example, peptides, which containamino acids, some of which are hydrophobic and some of which arehydrophilic. Aside from peptides, carbohydrates may be used.

Peptides having as few as two amino-acid residues may be used,preferably two to ten residues, if also coupled to other moieties suchas chelating agents. The linker should be a low molecular weightconjugate, preferably having a molecular weight of less than 50,000daltons, and advantageously less than about 20,000 daltons, 10,000daltons or 5,000 daltons, including the metal ions in the chelates. Forinstance, the known peptide DTPA-Tyr-Lys(DTPA)-OH (wherein DTPA isdiethylenetriaminepentaacetic acid) has been used to generate antibodiesagainst the indium-DTPA portion of the molecule. However, by use of thenon-indium-containing molecule, and appropriate screening steps, new Absagainst the tyrosyl-lysine dipeptide can be made. More usually, theantigenic peptide will have four or more residues, such as the peptideDOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂, wherein DOTA is1,4,7,10-tetraazacyclododecanetetraacetic acid and HSG is the histaminesuccinyl glycyl group of the formula:

The non-metal-containing peptide may be used as an immunogen, withresultant Abs screened for reactivity against the Phe-Lys-Tyr-Lysbackbone.

The invention also contemplates the incorporation of unnatural aminoacids, e.g., D-amino acids, into the backbone structure to ensure that,when used with the final bsAb/linker system, the arm of the bsAb whichrecognizes the linker moiety is completely specific. The inventionfurther contemplates other backbone structures such as those constructedfrom non-natural amino acids and peptoids.

The peptides to be used as immunogens are synthesized conveniently on anautomated peptide synthesizer using a solid-phase support and standardtechniques of repetitive orthogonal deprotection and coupling. Freeamino groups in the peptide, that are to be used later for chelateconjugation, are advantageously blocked with standard protecting groupssuch as an acetyl group. Such protecting groups will be known to theskilled artisan. See Greene and Wuts Protective Groups in OrganicSynthesis, 1999 (John Wiley and Sons, N.Y.). When the peptides areprepared for later use within the bsAb system, they are advantageouslycleaved from the resins to generate the corresponding C-terminal amides,in order to inhibit in vivo carboxypeptidase activity.

The haptens of the immunogen comprise an immunogenic recognition moiety,for example, a chemical hapten. Using a chemical hapten, preferably theHSG hapten, high specificity of the linker for the antibody isexhibited. This occurs because antibodies raised to the HSG hapten areknown and can be easily incorporated into the appropriate bispecificantibody. Thus, binding of the linker with the attached hapten would behighly specific for the antibody or antibody fragment.

Chelate Moieties

The presence of hydrophilic chelate moieties on the linker moietieshelps to ensure rapid in vivo clearance. In addition to hydrophilicity,chelators are chosen for their metal-binding properties, and are changedat will since, at least for those linkers whose bsAb epitope is part ofthe peptide or is a non-chelate chemical hapten, recognition of themetal-chelate complex is no longer an issue.

Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with ⁴⁷Sc, ⁵²Fe, ⁵⁵Co, ⁶⁷Ga,⁶⁸Ga, ¹¹¹In, ⁸⁹Zr, ⁹⁰Y, ¹⁶¹Th, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, and ²²⁵Ac forradio-imaging and RAIT. The same chelators, when complexed withnon-radioactive metals, such as Mn, Fe and Gd can be used for MRI, whenused along with the bsAbs of the invention. Macrocyclic chelators suchas NOTA (1,4,7-triaza-cyclononane-N,N′,N″-triacetic acid), DOTA, andTETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are ofuse with a variety of metals and radiometals, most particularly withradionuclides of Ga, Y and Cu, respectively.

DTPA and DOTA-type chelators, where the ligand includes hard basechelating functions such as carboxylate or amine groups, are mosteffective for chelating hard acid cations, especially Group IIa andGroup IIIa metal cations. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelators such as macrocyclic polyethers are of interest forstably binding nuclides such as ²²³Ra for RAIT. Porphyrin chelators maybe used with numerous radiometals, and are also useful as certain coldmetal complexes for bsAb-directed immuno-phototherapy. More than onetype of chelator may be conjugated to a carrier to bind multiple metalions, e.g., cold ions, diagnostic radionuclides and/or therapeuticradionuclides. Particularly useful therapeutic radionuclides include,but are not limited to, ³²P, ³³P, ⁴⁷SC, ⁶⁴CU, ⁶⁷Cu, ⁶⁷Ga, ⁹⁰Y, ¹¹¹Ag,¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷L, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra and ²²⁵Ac. Particularlyuseful diagnostic/detection radionuclides include, but are not limitedto, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ^(94m)TC, ⁹⁴Tc,⁹⁹ mTc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd and ¹⁷⁵Lu.

Chelators such as those disclosed in U.S. Pat. No. 5,753,206, especiallythiosemi-carbazonylglyoxylcysteine (Tscg-Cys) andthiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelators areadvantageously used to bind soft acid cations of Tc, Re, Bi and othertransition metals, lanthanides and actinides that are tightly bound tosoft base ligands, especially sulfur- or phosphorus-containing ligands.It can be useful to link more than one type of chelator to a peptide,e.g., a DTPA or similar chelator for, say In(III) cations, and athiol-containing chelator, e.g., Tscg-Cys, for Tc cations. Becauseantibodies to a di-DTPA hapten are known (Barbet '395, supra) and arereadily coupled to a targeting antibody to form a bsAb, it is possibleto use a peptide hapten with cold diDTPA chelator and another chelatorfor binding a radioisotope, in a pretargeting protocol, for targetingthe radioisotope. One example of such a peptide isAc-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys-)-NH₂. This peptide can bepreloaded with ln(III) and then labeled with 99-m-Tc cations, theIn(III) ions being preferentially chelated by the DTPA and the Tccations binding preferentially to the thiol-containing Tscg-Cys. Otherhard acid chelators such as NOTA, DOTA, TETA and the like can besubstituted for the DTPA groups, and Mabs specific to them can beproduced using analogous techniques to those used to generate theanti-di-DTPA Mab.

It will be appreciated that two different hard acid or soft acidchelators can be incorporated into the linker, e.g., with differentchelate ring sizes, to bind preferentially to two different hard acid orsoft acid cations, due to the differing sizes of the cations, thegeometries of the chelate rings and the preferred complex ion structuresof the cations. This will permit two different metals, one or both ofwhich may be radioactive or useful for MRI enhancement, to beincorporated into a linker for eventual capture by a pretargeted bsAb.

Preferred chelators include NOTA, DOTA and Tscg and combinationsthereof. These chelators have been incorporated into a chelator-peptideconjugate motif as exemplified in the following constructs:

(a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;

(b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;

(c) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

The chelator-peptide conjugates (d) and (e), above, has been shown tobind ⁶⁸Ga and is thus useful in positron emission tomography (PET)applications.

Chelators are coupled to the linker moieties using standard chemistrieswhich are discussed more fully in the working Examples below. Briefly,the synthesis of the peptideAc-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)-NH₂ was accomplished by firstattaching Aloc-Lys(Fmoc)-OH to a Rink amide resin on the peptidesynthesizer. The protecting group abbreviations “Aloc” and “Fmoc” usedherein refer to the groups allyloxycarbonyl and fluorenylmethyloxycarbonyl. The Fmoc-Cys(Trt)-OH and TscG were then added to the sidechain of the lysine using standard Fmoc automated synthesis protocols toform the following peptide: Aloc-Lys(Tscg-Cys(Trt)-rink resin. The Alocgroup was then removed. The peptide synthesis was then continued on thesynthesizer to make the following peptide:(Lys(Aloc)-D-Tyr-Lys(Aloc)-Lys(Tscg-Cys(Trt)-)-rink resin. FollowingN-terminus acylation, and removal of the side chain Aloc protectinggroups. The resulting peptide was then treated with activatedN-trityl-HSG-OH until the resin gave a negative test for amines usingthe Kaiser test. See Karacay et al. Bioconjugate Chem. 11:842-854(2000). The synthesis of Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)-NH₂,as well as the syntheses of DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; andDOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ are described in greater detailbelow.

Preparation of Metal Chelates

Chelator-peptide conjugates may be stored for long periods as solids.They may be metered into unit doses for metal-binding reactions, andstored as unit doses either as solids, aqueous or semi-aqueoussolutions, frozen solutions or lyophilized preparations. They may belabeled by well-known procedures. Typically, a hard acid cation isintroduced as a solution of a convenient salt, and is taken up by thehard acid chelator and possibly by the soft acid chelator. However,later addition of soft acid cations leads to binding thereof by the softacid chelator, displacing any hard acid cations which may be chelatedtherein. For example, even in the presence of an excess of cold¹¹¹InCl₃, labeling with 99m-Tc(V) glucoheptonate or with Tc cationsgenerated in situ with stannous chloride and Na99m-TcO₄ proceedsquantitatively on the soft acid chelator. Other soft acid cations suchas ¹⁸⁶Re, ¹⁸⁸Re, ²¹³Bi and divalent or trivalent cations of Mn, Co, Ni,Pb, Cu, Cd, Au, Fe, Ag (monovalent), Zn and Hg, especially ⁶⁴Cu and⁶⁷Cu, and the like, some of which are useful for radioimmunodiagnosis orradioimmunotherapy, can be loaded onto the linker peptide by analogousmethods. Re cations also can be generated in situ from perrhenate andstannous ions or a prereduced rhenium glucoheptonate or othertranschelator can be used. Because reduction of perrhenate requires morestannous ion (typically above 200 μg/mL final concentration) than isneeded for the reduction of Tc, extra care needs to be taken to ensurethat the higher levels of stannous ion do not reduce sensitive disulfidebonds such as those present in disulfide-cyclized peptides. Duringradiolabeling with rhenium, similar procedures are used as are used withthe Tc-99m. A preferred method for the preparation of ReO metalcomplexes of the Tscg-Cys-ligands is by reacting the peptide withReOCl₃(P(Ph₃)₂ but it is also possible to use other reduced species suchas ReO(ethylenediamine)-2

VI. Methods of Administration

It should be noted that much of the discussion presented herein belowfocuses on the use of the inventive bispecific antibodies and targetableconstructs in the context of treating diseased tissue. The inventioncontemplates, however, the use of the inventive bispecific antibodiesand targetable constructs in treating and/or imaging tissue and organsusing the methods described in U.S. Pat. Nos. 6,126,916; 6,077,499;6,010,680; 5,776,095; 5,776,094; 5,776,093; 5,772,981; 5,753,206;5,746,996; 5,697,902; 5,328,679; 5,128,119; 5,101,827; and 4,735,210, solong as there is expression of CSAp in said tissues and organs. As usedherein, the term “tissue” refers to diseased tissues, more specificallymalignant tissues expressing CSAp, including but not limited to,malignant tissues of the colon, rectum, pancreas, and ovary.

The administration of a bsAb and a therapeutic agent associated with thelinker moieties discussed above may be conducted by administering thebsAb at some time prior to administration of the therapeutic agent whichis associated with the linker moiety. The doses and timing of thereagents can be readily devised by a skilled artisan, and are dependenton the specific nature of the reagents employed. If a bsAb-F(ab′)₂derivative is given first, then a waiting time of 24-72 hr beforeadministration of the linker moiety would be appropriate. If an IgG-Fab′bsAb conjugate is the primary targeting vector, then a longer waitingperiod before administration of the linker moiety would be indicated, inthe range of 3-10 days.

As used herein, the term “therapeutic agent” includes, but is notlimited to a drug, prodrug and/or toxin. The terms “drug,” “prodrug,”and “toxin” are defined throughout the specification. A diagnostic agentis more often used to determine the kind of disease present, while adetection agent is more often used for localization and diagnosis.However, as described herein, the term diagnostic agent can also be usedto refer to a detection agent.

After sufficient time has passed for the bsAb to target to the diseasedtissue, the diagnostic/detection agent is administered. Subsequent toadministration of the diagnostic/detection agent, imaging can beperformed. Tumors can be detected in body cavities by means of directlyor indirectly viewing various structures to which energy of theappropriate wavelength is delivered and then collected. Lesions at anybody site can be viewed so long as nonionizing radiation or energy canbe delivered and recaptured from these structures. For example, PETwhich is a high resolution, non-invasive, imaging technique can be usedwith the inventive antibodies for the visualization of human disease. InPET, 511 keV gamma photons produced during positron annihilation decayare detected.

The invention generally contemplates the use of diagnostic/detectionagents which emit 25-600 keV gamma particles and/or positrons. Examplesof such agents include, but are not limited to ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu,⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹¹¹In, ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd and ¹⁷⁵Lu.

The present antibodies or antibody fragments can be used in a method ofphotodynamic therapy (PDT) as discussed in U.S. Pat. Nos. 6,096,289;4,331,647; 4,818,709; 4,348,376; 4,361,544; 4,444,744; 5,851,527.

In PDT, a photosensitizer, e.g., a hematoporphyrin derivative such asdihematoporphyrin ether, is administered to a subject. Anti-tumoractivity is initiated by the use of light, e.g., 630 nm. Alternatephotosensitizers can be utilized, including those useful at longerwavelengths, where skin is less photosensitized by the sun. Examples ofsuch photosensitizers include, but are not limited to, benzoporphyrinmonoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminumphthalocyanine (AlSPc) and lutetium texaphyrin (Lutex).

Additionally, in PDT, a diagnostic agent is injected, for example,systemically, and laser-induced fluorescence can be used by endoscopesto detect sites of cancer which have accreted the light-activated agent.For example, this has been applied to fluorescence bronchoscopicdisclosure of early lung tumors. Doiron et al Chest 76:32 (1979). Inanother example, the antibodies and antibody fragments can be used insingle photon emission. For example, a Tc-99m-labeleddiagnostic/detection agent can be administered to a subject followingadministration of the inventive antibodies or antibody fragments. Thesubject is then scanned with a gamma camera which produces single-photonemission computed tomographic images and defines the lesion or tumorsite.

Therapeutically useful immunoconjugates can be obtained by conjugatingphotoactive agents or dyes to an antibody composite. Fluorescent andother chromogens, or dyes, such as porphyrins sensitive to visiblelight, have been used to detect and to treat lesions by directing thesuitable light to the lesion. In therapy, this has been termedphotoradiation, phototherapy, or photodynamic therapy (Jori et al.(eds.), Photodynamic Therapy of Tumors and Other Diseases (LibreriaProgetto 1985); van den Bergh, Chem. Britain 22:430 (1986)). Moreover,monoclonal antibodies have been coupled with photoactivated dyes forachieving phototherapy. Mew et al., J. Immunol. 130:1473 (1983); idem.,Cancer Res. 45:4380 (1985); Oseroff et al., Proc. Natl. Acad. Sci. USA83:8744 (1986); idem., Photochem. Photobiol. 46:83 (1987); Hasan et al,Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med.9:422 (1989); Pelegrin et al., Cancer 67:2529 (1991). However, theseearlier studies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes.

The linker moiety may also be conjugated to an enzyme capable ofactivating a prodrug at the target site or improving the efficacy of anormal therapeutic by controlling the body's detoxification pathways.Following administration of the bsAb, an enzyme conjugated to the linkermoiety, a low MW hapten recognized by the second arm of the bsAb, isadministered. After the enzyme is pretargeted to the target site, acytotoxic drug is injected, which is known to act at the target site.The drug may be one which is detoxified by the mammal's ordinarydetoxification processes. For example, the drug may be converted intothe potentially less toxic glucuronide in the liver. The detoxifiedintermediate can then be reconverted to its more toxic form by thepretargeted enzyme at the target site. Alternatively, an administeredprodrug can be converted to an active drug by the pretargeted enzyme.The pretargeted enzyme improves the efficacy of the treatment byrecycling the detoxified drug. This approach can be adopted for use withany enzyme-drug pair.

Certain cytotoxic drugs that are useful for anticancer therapy arerelatively insoluble in serum. Some are also quite toxic in anunconjugated form, and their toxicity is considerably reduced byconversion to prodrugs. Conversion of a poorly soluble drug to a moresoluble conjugate, e.g., a glucuronide, an ester of a hydrophilic acidor an amide of a hydrophilic amine, will improve its solubility in theaqueous phase of serum and its ability to pass through venous, arterialor capillary cell walls and to reach the interstitial fluid bathing thetumor. Cleavage of the prodrug deposits the less soluble drug at thetarget site. Many examples of such prodrug-to-drug conversions aredisclosed in Hansen U.S. Pat. No. 5,851,527, incorporated herein in itsentirety.

Conversion of certain toxic substances such as aromatic or alicyclicalcohols, thiols, phenols and amines to glucuronides in the liver is thebody's method of detoxifying them and making them more easily excretedin the urine. One type of antitumor drug that can be converted to such asubstrate is epirubicin, a 4-epimer of doxorubicin (Adriamycin), whichis an anthracycline glycoside and has been shown to be a substrate forhuman beta-D-glucuronidase See, e.g., Arcamone Cancer Res. 45:5995(1985). Other analogues with fewer polar groups are expected to be morelipophilic and show greater promise for such an approach. Other drugs ortoxins with aromatic or alicyclic alcohol, thiol or amine groups arecandidates for such conjugate formation. These drugs, or other prodrugforms thereof, are suitable candidates for the site-specific enhancementmethods of the present invention.

The prodrug CPT-11 (irinotecan) is converted in vivo by carboxylesteraseto the active metabolite SN-38. One application of the invention,therefore, is to use a bsAb targeted against a tumor and a hapten (e.g.di-DTPA) followed by injection of a di-DTPA-carboxylesterase conjugate.Once a suitable tumor-to-background localization ratio has beenachieved, the CPT-11 is given and the tumor-localized carboxylesteraseserves to convert CPT-11 to SN-38 at the tumor. Due to its poorsolubility, the active SN-38 will remain in the vicinity of the tumorand, consequently, will exert an effect on adjacent tumor cells that arenegative for the antigen being targeted. This is a fiurer advantage ofthe method. Modified forms of carboxylesterases have been described andare within the scope of the invention. See, e.g., Potter et al., CancerRes. 58:2646-2651 (1998) and Potter et al., Cancer Res. 58:3627-3632(1998).

Etoposide is a widely used cancer drug that is detoxified to a majorextent by formation of its glucuronide and is within the scope of theinvention. See, e.g., Hande et al., Cancer Res. 48:1829-1834 (1988).Glucuronide conjugates can be prepared from cytotoxic drugs and can beinjected as therapeutics for tumors pre-targeted with mAb-glucuronidaseconjugates. See, e.g., Wang et al., Cancer Res. 52:4484-4491 (1992).Accordingly, such conjugates also can be used with the pre-targetingapproach described here. Similarly, designed prodrugs based onderivatives of daunomycin and doxorubicin have been described for usewith carboxylesterases and glucuronidases. See, e.g., Bakina et al., JMed. Chem. 40:4013-4018 (1997). Other examples of prodrug/enzyme pairsthat can be used within the present invention include, but are notlimited to, glucuronide prodrugs of hydroxy derivatives of phenolmustards and beta-glucuronidase; phenol mustards or CPT-11 andcarboxypeptidase; methotrexate-substituted alpha-amino acids andcarboxypeptidase A; penicillin or cephalosporin conjugates of drugs suchas 6-mercaptopurine and doxorubicin and beta-lactamase; etoposidephosphate and alkaline phosphatase.

The enzyme capable of activating a prodrug at the target site orimproving the efficacy of a normal therapeutic by controlling the body'sdetoxification pathways may alternatively be conjugated to the hapten.The enzyme-hapten conjugate is administered to the subject followingadministration of the pre-targeting bsAb and is directed to the targetsite. After the enzyme is localized at the target site, a cytotoxic drugis injected, which is known to act at the target site, or a prodrug formthereof which is converted to the drug in situ by the pretargetedenzyme. As discussed above, the drug is one which is detoxified to forman intermediate of lower toxicity, most commonly a glucuronide, usingthe mammal's ordinary detoxification processes. The detoxifiedintermediate, e.g., the glucuronide, is reconverted to its more toxicform by the pretargeted enzyme and thus has enhanced cytotoxicity at thetarget site. This results in a recycling of the drug. Similarly, anadministered prodrug can be converted to an active drug through normalbiological processess. The pretargeted enzyme improves the efficacy ofthe treatment by recycling the detoxified drug. This approach can beadopted for use with any enzyme-drug pair.

The invention further contemplates the use of the inventive bsAb and thediagnostic agent(s) in the context of Boron Neutron Capture Therapy(BNCT) protocols. BNCT is a binary system designed to deliver ionizingradiation to tumor cells by neutron irradiation of tumor-localized ¹⁰Batoms. BNCT is based on the nuclear reaction which occurs when a stableisotope, isotopically enriched ¹⁰B (present in 19.8% natural abundance),is irradiated with thermal neutrons to produce an alpha particle and a⁷Li nucleus. These particles have a path length of about one celldiameter, resulting in high linear energy transfer. Just a few of theshort-range 1.7 MeV alpha particles produced in this nuclear reactionare sufficient to target the cell nucleus and destroy it. Success withBNCT of cancer requires methods for localizing a high concentration of¹⁰B at tumor sites, while leaving non-target organs essentiallyboron-free. Compositions and methods for treating tumors in subjectsusing pre-targeting bsAb for BNCT are described in co-pending patentapplication Ser. No. 09/205,243, incorporated herein in its entirety andcan easily be modified for the purposes of the present invention.

It should also be noted that a bispecific antibody or antibody fragmentcan be used in the present method, with at least one binding sitespecific to an antigen at a target site and at least one other bindingsite specific to the enzyme component of the antibody-enzyme conjugate.Such an antibody can bind the enzyme prior to injection, therebyobviating the need to covalently conjugate the enzyme to the antibody,or it can be injected and localized at the target site and, afternon-targeted antibody has substantially cleared from the circulatorysystem of the mammal, enzyme can be injected in an amount and by a routewhich enables a sufficient amount of the enzyme to reach a localizedantibody or antibody fragment and bind to it to form the antibody-enzymeconjugate in situ.

It should also be noted that the invention also contemplates the use ofmultivalent target-binding proteins which have at least three differenttarget binding sites as described in patent application Ser. No.09/911,610, and is incorporated herein by reference. Multivalentantibodies have been made by cross-linking several Fab-like fragmentsvia chemical linkers. See U.S. Pat. Nos. 5,262,524; 5,091,542 andLandsdorp et al., Europ. J. Immunol. 16: 679-83 (1986). Multivalentantibodies also have been made by covalently linking several singlechain Fv molecules (scFv) to form a single polypeptide. See U.S. Pat.No. 5,892,020. A multivalent antibody which is basically an aggregate ofscFv molecules has been disclosed in U.S. Pat. Nos. 6,025,165 and5,837,242. A trivalent target binding protein comprising three scFvmolecules has been described in Krott et al. Protein Engineering 10(4):423-433 (1997).

A clearing agent may be used which is given between doses of the bsAband the linker moiety. The present inventors have discovered that aclearing agent of novel mechanistic action may be used with theinvention, namely a glycosylated anti-idiotypic (anti-Id) Fab′ fragmenttargeted against the disease targeting arm(s) of the bsAb. For example,anti-CSAp (Mu-9 Ab)×anti-peptide bsAb is given and allowed to accrete indisease targets to its maximum extent. To clear residual bsAb, ananti-idiotypic (anti-Id) Ab to Mu-9 is given, preferably as aglycosylated Fab′ fragment. The clearing agent binds to the bsAb in amonovalent manner, while its appended glycosyl residues direct theentire complex to the liver, where rapid metabolism takes place. Thenthe therapeutic which is associated with the linker moiety is given tothe subject. The anti-Id Ab to the Mu-9 arm of the bsAb has a highaffinity and the clearance mechanism differs from other disclosedmechanisms (see Goodwin et al., ibid), as it does not involvecross-linking, because the anti-Id-Fab′ is a monovalent moiety.

VII. Pharmaceutically Suitable Excipient

The humanized, chimeric or human anti-CSAp antibodies and fragmentsthereof to be delivered to a subject can consist of the antibody alone,immunoconjugate, fusion protein, or can comprise one or morepharmaceutically suitable excipients, one or more additionalingredients, or some combination of these. Preferably, the anti-CSApantibody is a Mu-9 antibody.

The Mu-9 immunoconjugate, naked antibody, fusion protein, and fragmentsthereof of the present invention can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby theimmunoconjugate or naked antibody is combined in a mixture with apharmaceutically suitable excipient. Sterile phosphate-buffered salineis one example of a pharmaceutically suitable excipient. Other suitableexcipients are well known to those in the art. See, for example, Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The immunoconjugate, naked antibody, fusion protein, and fragmentsthereof of the present invention can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic conjugate or nakedantibody. Control release preparations can be prepared through the useof polymers to complex or adsorb the immunoconjugate or naked antibody.For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate, antibody fusion proteins, or naked antibody mayalso be administered to a mammal subcutaneously or even by otherparenteral routes. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. In general, the dosage of anadministered immunoconjugate, fusion protein or naked antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of immunoconjugate, antibody fusion protein or naked antibodythat is in the range of from about 1 mg/kg to 20 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths, or even less frequently, as may be the case forradioimmunoconjugates. The dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

For purposes of therapy, the immunoconjugate, fusion protein, or nakedantibody, and fragments thereof are administered to a subject in atherapeutically effective amount. A suitable subject for the presentinvention is a mammal, preferably a human but a non-human mammal such asa dog, cat or horse is also contemplated. An antibody preparation issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient mammal.

For diagnostic purposes, the immunoconjugate, fusion protein, or nakedantibody, and fragments thereof are administered to a subject in adiagnostically effective amount. An antibody preparation is said to beadministered in a “diagnostically effective amount” if the amountadministered is generally sufficient to diagnose or detect a condition,malignancy, disease or disorder in a subject, usually without anypharmacological effects on the host.

VIII. Expression Vectors

The present invention also embraces nucleic acids that encode chimeric,humanized or human anti-CSAp antibodies, fusion proteins and fragmentsthereof. Expression vectors that comprise such nucleic acids also areincluded in the invention. The DNA sequence encoding a humanized,chimeric or human Mu-9 antibody can be recombinantly engineered into avariety of known host vectors that provide for replication of thenucleic acid. These vectors can be designed, using known methods, tocontain the elements necessary for directing transcription, translation,or both, of the nucleic acid in a cell to which it is delivered. Knownmethodology can be used to generate expression constructs that have aprotein-coding sequence operably linked with appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques and synthetic techniques. For example,see Sambrook et al, 1989, MOLECULAR CLONING: A LABORATORY MANUAL, ColdSpring Harbor Laboratory (New York); Ausubel et al., 1997, CURRENTPROTOCOLS 1N MOLECULAR BIOLOGY, John Wiley & Sons (New York). Alsoprovided for in this invention is the delivery of a polynucleotide notassociated with a vector.

Vectors suitable for use in the instant invention can be viral ornon-viral. Particular examples of viral vectors include adenovirus, AAV,herpes simplex virus, lentivirus, and retrovirus vectors. An example ofa non-viral vector is a plasmid. In a preferred embodiment, the vectoris a plasmid.

An expression vector, as described herein, is a polynucleotidecomprising a gene that is expressed in a host cell. Typically, geneexpression is placed under the control of certain regulatory elements,including constitutive or inducible promoters, tissue-specificregulatory elements, and enhancers. Such a gene is said to be “operablylinked to” the regulatory elements.

Preferably, the expression vector of the instant invention comprises theDNA sequence encoding a humanized, chimeric or human Mu-9 antibody,which includes both the heavy and the light chain variable and constantregions. However, two expression vectors may be used, with onecomprising the heavy chain variable and constant regions and the othercomprising the light chain variable and constant regions. Stillpreferred, the expression vector further comprises a promoter, a DNAsequence encoding a secretion signal peptide, a genomic sequenceencoding a human IgG1 heavy chain constant region, an Ig enhancerelement and at least one DNA sequence encoding a selection marker.

The representative embodiments described below are simply used toillustrate the invention. Those skilled in these arts will recognizethat variations of the present materials fall within the broad genericscope of the claimed invention. The contents of all references mentionedherein are incorporated by reference.

IX. EXAMPLES Example 1 Synthesis ofAc-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)-NH₂ (IMP 243)

The peptide was synthesized as described by Karacay et. al. BioconjugateChem. 11:842-854 (2000) except D-tyrosine was used in place of theL-tyrosine and the N-trityl-HSG-OH was used in place of the DTPA. Thefinal coupling of the N-trityl-HSG-OH was carried out using a ten foldexcess of N-trityl-HSG-OH relative to the peptide on the resin. TheN-trityl-HSG-OH (0.28 M in NMP) was activated using one equivalent(relative to HSG) of N-hydroxybenzotriazole, one equivalent ofbenzotrazole-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate (BOP) and two equivalents of diisopropylethylamine.The activated substrate was mixed with the resin for 15 hr at roomtemperature.

Example 2 Tc-99m Kit Formulation Comprising IMP 243

A formulation buffer was prepared which contained 22.093 ghydroxypropyl-β-cyclodextrin, 0.45 g 2,4-dihydroxybenzoic acid, 0.257 gacetic acid sodium salt, and 10.889 g α-D-glucoheptonic acid sodium saltdissolved in 170 mL nitrogen degassed water. The solution was adjustedto pH 5.3 with a few drops of 1 M NaOH then further diluted to a totalvolume of 220 mL. A stannous buffer solution was prepared by diluting0.2 mL of SnCl₂ (200 mg/ml) with 3.8 mL of the formulation buffer. Thepeptide Ac-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)-NH₂ (0.0026 g), wasdissolved in 78 mL of the buffer solution and mixed with 0.52 mL of thestannous buffer. The peptide solution was then filtered through a 0.22□m Millex GV filter in 1.5 mL aliquots into 3 mL lyophilization vials.The filled vials were frozen immediately, lyophilized and crimp sealedunder vacuum.

Pertechnetate solution (27 mCi) in 1.5 mL of saline was added to thekit. The kit was incubated at room temperature for 10 min and heated ina boiling water bath for 25 min. The kit was cooled to room temperaturebefore use.

Example 3 Peptides for Carrying Therapeutic/Imaging Radioisotopes toTumors via Bispecific Antibody Tumor Pretargeting

DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ (IMP 237) was synthesized to delivertherapeutic radioisotopes such as ⁹⁰Y or ¹⁷⁷Lu to tumors via bispecificantibody tumor pretargeting. The bispecific antibody is composed of oneportion which binds to an antigen on the tumor and another portion whichbinds to the HSG peptide. The antibody which binds the HSG peptide is679. This system can also be used to deliver imaging isotopes such as¹¹¹In-111.

Synthesis of IMP 237

IMP 237 was synthesized on Sieber Amide resin (Nova-Biochem) usingstandard Fmoc based solid phase peptide synthesis to assemble thepeptide backbone with the following protected amino acids, in order:Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(But)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Phe-OH,(Reagents from Advanced Chemtech) tri-t-butyl DOTA (Macrocyclics). Theside lysine side chains were then deprotected with Pd[P(Ph)₃]₄ by themethod of Dangles et. al. J. Org. Chem. 52:4984-4993 (1987). The HSGligands were then added as Trityl HSG (synthesis described below) usingthe BOP/HBTU double coupling procedure used to attach the amino acids.The peptide was cleaved from the resin and the protecting groups wereremoved by treatment with TFA. The peptide was purified by HPLC toafford 0.6079 g of peptide from 1.823 g ofFmoc-Lys(Aloc)-Tyr(But)-Lys(Aloc)-NH-Sieber amide resin.

Synthesis of N-Trityl-HSG-OH

Glycine t-butyl ester hdyrochloride (15.263 g, 9.1×10⁻² mol) and 19.760g Na₂CO₃ were mixed, then suspended in 50 mL H₂O and cooled in an icebath. Succinic anhydride (9.142 g, 9.14×10⁻² mol) was then added to thereaction solution which was allowed to warm slowly to room temperatureand stir for 18 hr. Citric acid (39.911 g) was dissolved in 50 mL H₂Oand slowly added to the reaction solution and then extracted with 2×150mL EtOAc. The organic extracts were dried over Na₂SO₄, filtered andconcentrated to afford 25.709 g of a white solid.

The crude product (25.709 g) was dissolved in 125 mL dioxane, cooled ina room temperature water bath and mixed with 11.244 g ofN-hydroxysuccinimide. Diisopropylcarbodiimide 15.0 mL was added to thereaction solution which was allowed to stir for one hour. Histaminedihydrochloride (18.402 g, 1.00×10⁻¹ mol) was then dissolved in 100 mLDMF and 35 mL diisopropylethylamine. The histamine mixture was added tothe reaction solution which was stirred at room temperature for 21 hr.The reaction was quenched with 100 mL water and filtered to remove aprecipitate. The solvents were removed under hi-vacuum on the rotaryevaporator. The crude product was dissolved in 300 mL dichloromethaneand extracted with 100 mL saturated NaHCO₃. The organic layer was driedover Na₂SO₄ and concentrated to afford 34.19 g of crude product as ayellow oil.

The crude product (34.19 g) was dissolved in 50 mL chloroform and mixedwith

31 mL diisopropylethylamine. Triphenylmethyl chloride (25.415 g) wasdissolved in 50 ml chloroform and added dropwise to the stirred reactionsolution which was cooled in an ice bath. The reaction was stirred for45 min and then quenched with 100 mL H₂O. The layers were separated andthe organic solution was dried over Na₂SO₄ and concentrated to form agreen gum. The gum was triturated with 100 mL Et₂O to form a yellowprecipitate which was washed with 3×50 mL portions of Et₂O. The solidwas vacuum dried to afford 30.641 g (59.5% overall yield) ofN-trityl-HSG-t-butyl ester.

N-trityl-HSG-t-butyl ester (20.620 g, 3.64×10⁻² mol) was dissolved in asolution of 30 mL chloroform and 35 mL glacial acetic acid. The reactionwas cooled in an ice bath and 15 mL of BF₃-Et₂O was slowly added to thereaction solution. The reaction was allowed to warm slowly to roomtemperature and mix for 5 hr. The reaction was quenched by pouring into200 mL 1M NaOH and the product was extracted with 200 mL chloroform. Theorganic layer was dried over Na₂SO₄ and concentrated to afford a crudegum which was triturated with 100 mL Et₂O to form a precipitate. Thecrude precipitate was poured into 400 mL 0.5 M pH 7.5 phosphate bufferand extracted with 2×200 mL EtOAc. The aqueous layer was acidified to pH3.5 with 1 M HCl and extracted with 2×200 mL chloroform. A precipitateformed and was collected by filtration (8.58 g). The precipitate was thedesired product by HPLC comparison to a previous sample (ESMS MH+ 511).

Radiolabeling

⁹⁰Y Kit Preparation

DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ was dissolved in 0.25 M NH₄OAc/10%HPCD buffer at concentrations of 9, 18, 35, 70 and 140 μg/mL. Thesolutions were sterile filtered through a 0.22 μm Millex GV filter inone mL aliquots into acid washed lyophilization vials. The filled vialswere frozen immediately on filling and lyophilized. When thelyophilization cycle was complete the vials were sealed under vacuum andcrimp sealed upon removal from the lyophilizer.

The ⁹⁰Y (˜400 MC1/kit) was diluted to 1 mL in deionized water and addedto the lyophilized kits. The kits were heated in a boiling water bathfor 15 min, the vials were cooled to room temperature and the labeledpeptides were evaluated by reverse phase HPLC(HPLC conditions: WatersNova-Pak C-18, 8×100 mm RCM column eluted at 3 mL/min with a lineargradient from 100% (0.1% TFA in H₂O) to 100% (90% CH₃CN, 0.1% TFA, 10%H₂O)). The HPLC analysis revealed that the minimum concentration ofpeptide needed for complete labeling, with this formulation, was 35μg/mL. The reverse phase HPLC trace showed a sharp ⁹⁰Y labeled peptidepeak. The labeled peptide was completely bound when mixed with excess679 IgG by size exclusion HPLC.

Labeling with ¹¹¹In

The ¹¹¹In (˜300 μCi/kit) was diluted to 0.5 mL in deionized water andadded to the lyophilized kits. The kits were heated in a boiling waterbath for 15 min, the vials were cooled and 0.5 mL of 2.56×10⁻⁵ M In in0.5 M acetate buffer was added and the kits were again heated in theboiling water bath for 15 min. The labeled peptide vials were cooled toroom temperature and evaluated by reverse phase HPLC(HPLC conditions:Waters Nova-Pak C-18, 8×100 mm RCM column eluted at 3 mL/min with alinear gradient from 100% (0.1% TFA in H₂O) to 100% (90% CH₃CN, 0.1%TFA, 10% H₂O)). The HPLC analysis revealed that the minimumconcentration of peptide needed for labeling (4.7% loose ¹¹¹In), withthis formulation, was 35 μg/mL. The reverse phase HPLC trace showed asharp ¹¹¹In labeled peptide peak. The labeled peptide was completelybound when mixed with excess 679 IgG by size exclusion HPLC.

In-Vivo Studies

Nude mice bearing GW-39 human colonic xenograft tumors (100-500 mg) wereinjected with the bispecific antibody hMN-14×m679 (1.5×10⁻¹⁰ mol). Theantibody was allowed to clear for 24 hr before the ¹¹¹In labeled peptide(8.8 μCi, 1.5×10⁻¹¹ mol) was injected. The animals were sacrificed at 3,24, 48 hr post injection.

The results of the biodistribution studies of the peptide in the micepretargeted with hMN-14×m679 are shown in Table 1. The tumor tonon-tumor ratios of the peptides in the pretargeting study are show inTable 2.

TABLE 1 Pretargeting With ¹¹¹In Labeled Peptide 24 hr After Injection ofhMN-14 × m679 % Injected/g Tissue 3 hr After ¹¹¹In 24 hr After ¹¹¹In 48hr After ¹¹¹In Tissue IMP 237 IMP 237 IMP 237 GW-39 7.25 ± 2.79 8.38 ±1.70 5.39 ± 1.46 Liver 0.58 ± 0.13 0.62 ± 0.09 0.61 ± 0.16 Spleen 0.50 ±0.14 0.71 ± 0.16 0.57 ± 0.15 Kidney 3.59 ± 0.75 2.24 ± 0.40 1.27 ± 0.33Lungs 1.19 ± 0.26 0.44 ± 0.10 0.22 ± 0.06 Blood 2.42 ± 0.61 0.73 ± 0.170.17 ± 0.06 Stomach 0.18 ± 0.03 0.09 ± 0.02 0.07 ± 0.02 Sm. Int. 0.65 ±0.74 0.18 ± 0.03 0.11 ± 0.02 Lg. Int. 0.30 ± 0.07 0.17 ± 0.03 0.13 ±0.03

TABLE 2 Pretargeting With ¹¹¹In Labeled Peptides 24 hr After Injectionof hMN-14 × m679 Tumor/Non-Tumor Tissue Ratios 3 hr After ¹¹¹In 24 hrAfter ¹¹¹In 48 hr After ¹¹¹In Tissue IMP 237 IMP 237 IMP 237 Liver 12.6± 4.44 13.6 ± 2.83 8.88 ± 1.78 Spleen 15.1 ± 6.32 12.1 ± 2.86 9.50 ±1.62 Kidney 2.04 ± 0.74 3.84 ± 1.04 4.25 ± 0.19 Lungs 6.11 ± 1.96 19.6 ±5.91 25.4 ± 6.00 Blood 3.04 ± 1.13 11.9 ± 3.20 31.9 ± 4.79 Stomach 40.5± 16.5 104. ± 39.6 83.3 ± 16.5 Sm. Int. 18.9 ± 12.6 47.5 ± 10.3 49.5 ±7.83 Lg. Int. 25.2 ± 10.6 50.1 ± 16.7 43.7 ± 9.35

Serum Stability of DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ (IMP 237) andDOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂ (IMP 241)

Peptide Labeling and HPLC Analysis

The peptides, IMP 237 and IMP 241, were labeled according to theprocedure described by Karacay et. al. Bioconjugate Chem. 11:842-854(2000). The peptide, IMP 241 (0.0019 g), was dissolved in 587 μl 0.5 MNH₄Cl, pH 5.5. A 1.7 μL aliquot of the peptide solution was diluted with165 pt 0.5 M NH₄Cl, pH 5.5. The ¹¹¹In (1.8 mCi) in 10 μL was added tothe peptide solution and the mixture was heated in a boiling water bathfor 30 min.

The labeled peptide was analyzed by HPLC using a Waters 8×100 mmradial-pak, nova-pak C-18 RCM cartridge column. The column was eluted at3 mL/min with a linear gradient which started with 100% of 0.1% TFA inwater and went to 100% of 0.1% TFA in 90% acetonitrile and 10% waterover 10 min. There was about 6% loose ¹¹¹In in this labeling which cameout at the void volume of the column (1.6 min). There were also some¹¹¹In labeled peaks at 5 min and 6.6 to 8 min. The ¹¹¹In labeled peptidewas eluted at 8.8 min as a single peak. The HPLC profile of ¹¹¹In IMP237 was nearly identical to ¹¹¹In IMP 241.

Serum Stability

An aliquot (30 μL) of ¹¹¹In IMP 241 was placed in 300 μL of fresh mouseserum and placed in a 37° C. incubator. The peptide was monitored asdescribed above by HPLC.

An aliquot (24 μL) of ¹¹¹In IMP 237 was placed in 230 μL of fresh mouseserum and placed in a 37° C. incubator. The peptide was monitored asdescribed above by HPLC.

The analysis showed that the ¹¹¹In IMP 241 may have decomposed slightly(˜5%) after heating 22 hr in mouse serum at 37° C. The ¹¹¹In IMP 237 wasabout 70% converted to the shorter retention time peak after incubationfor 22 hr at 37° C.

CONCLUSION

The D-tyrosine in the IMP 241 peptide slows the decomposition of thepeptide in mouse serum compared to IMP 237.

In Vivo Stability of IMP 237 and IMP 241 Compared

The in vivo stabilities of ¹¹¹In IMP 237 and ¹¹¹In IMP 241 were comparedby examining (by HPLC) urine samples from mice at 30 and 60 min. Thepeptides, IMP 241 and IMP 237, were ¹¹¹In-111 labeled as describedabove.

The labeled peptides were injected into Balb/c mice which weresacrificed at 30 min and 60 min post injection of the peptides using onemouse per time point. The attached HPLC traces indicate that ¹¹¹In IMP241 was excreted intact while ¹¹¹In IMP 237 was almost completelymetabolized to a new ¹¹¹In labeled peptide.

CONCLUSION

The replacement of Tyr with D-Tyr in the peptide backbone minimizedmetabolism of the peptide in-vivo.

Additional In Vivo Studies

Nude mice bearing GW-39 human colonic xenograft tumors (100-500 mg) wereinjected with the bispecific antibody mMu9×m679 (1.5×10⁻¹⁰ mol). Theantibody was allowed to clear for 48 hr before the ¹¹¹In labeledpeptides (8.8 μCi, 1.5×10⁻¹¹ mol) were injected. The animals weresacrificed at 3, 24, 48 hr post injection.

The results of the biodistribution studies of the peptides in the micepretargeted with InMU9×m679 are shown in Table 3. The tumor to non-tumorratios of the peptides in the pretargeting study are show in Table 4.The data in Table 5 shows the biodistribution of the peptides in micethat were not pretreated with the bispecific antibody.

TABLE 3 Pretargeting With ¹¹¹In Labeled Peptides 48 hr After Injectionof mMU9 × m679 % Injected/g Tissue 3 hr After 24 hr After ¹¹¹In 48 hrAfter ¹¹¹In ¹¹¹In Peptide Peptide Peptide Tissue IMP 237 IMP 241 IMP 237IMP 241 IMP 237 IMP 241 GW-39 18.3 ± 7.17 26.7 ± 14.1 16.7 ± 8.22 14.8 ±4.56 12.9 ± 1.10 [12.3 ± 2.11  Liver 0.41 ± 0.10 0.66 ± 0.34 0.32 ± 0.080.32 ± 0.09 0.28 ± 0.09 0.32 ± 0.21 Spleen 0.34 ± 0.12 0.63 ± 0.38 0.34± 0.12 0.25 ± 0.07 0.28 ± 0.07 0.31 ± 0.22 Kidney 3.62 ± 0.71 4.28 ±0.77 2.51 ± 0.54 2.34 ± 0.70 1.78 ± 0.38 1.17 ± 0.43 Lungs 0.61 ± 0.151.03 ± 0.65 0.22 ± 0.07 0.21 ± 0.07 0.12 ± 0.04 0.14 ± 0.08 Blood 1.16 ±0.48 1.78 ± 1.49 0.21 ± 0.13 0.15 ± 0.05 0.08 ± 0.03 0.10 ± 0.09 Stomach0.12 ± 0.04 0.21 ± 0.09 0.05 ± 0.01 0.05 ± 0.02 0.04 ± 0.01 0.03 ± 0.02Sm. Int. 0.23 ± 0.04 0.50 ± 0.27 0.12 ± 0.02 0.09 ± 0.06 0.11 ± 0.080.07 ± 0.06 Lg. Int. 0.34 ± 0.16 0.38 ± 0.15 0.15 ± 0.07 0.10 ± 0.020.12 ± 0.07 0.09 ± 0.05

TABLE 4 Pretargeting With ¹¹¹In Labeled Peptides 48 hr After Injectionof mMU9 × m679 Tumor/Non-Tumor Tissue Ratios 3 hr After 24 hr After¹¹¹In 48 hr After ¹¹¹In ¹¹¹In Peptide Peptide Peptide Tissue IMP 237 IMP241 IMP 237 IMP 241 IMP 237 IMP 241 Liver 45.6 ± 17.8 41.8 ± 19.6 49.8 ±16.6 47.1 ± 8.68 49.1 ± 13.6 45.1 ± 13.9 Spleen 56.8 ± 23.8 43.5 ± 9.7747.4 ± 14.7 59.6 ± 13.0 47.5 ± 10.6 50.2 ± 19.0 Kidney 5.13 ± 2.18 6.05± 2.41 6.43 ± 2.24 6.58 ± 2.42 7.43 ± 1.02 11.2 ± 2.61 Lungs 30.5 ± 10.628.4 ± 12.8 76.4 ± 34.1 72.7 ± 21.9 115. ± 36.6 102. ± 37.1 Blood 18.6 ±12.0 19.0 ± 11.8 86.9 ± 36.2 108. ± 41.0 187. ± 76.3 181. ± 86.6 Stomach156. ± 86.1 126. ± 49.6 303. ± 95.9 328. ± 96.7 344. ± 101. 456. ± 193.Sm. Int. 80.7 ± 29.0 59.0 ± 31.0 143. ± 60.7 193. ± 83.7 153. ± 67.7217. ± 73.5 Lg. Int. 56.3 ± 19.7 78.6 ± 54.4 116. ± 36.9 155. ± 42.4133. ± 47.6 153. ± 43.1

TABLE 5 Biodistribution of ¹¹¹In Labeled Peptides Alone 30 min AfterIn-111 3 hr After In-111 24 hr After In-111 Peptide Peptide PeptideTissue IMP 237 IMP 241 IMP 237 IMP 241 IMP 237 IMP 241 GW-39 2.99 ± 1.112.73 ± 0.37 0.17 ± 0.05 0.31 ± 0.12 0.11 ± 0.02 0.11 ± 0.08 Liver 0.48 ±0.06 0.50 ± 0.09 0.15 ± 0.02 1.07 ± 1.61 0.15 ± 0.01 0.09 ± 0.04 Spleen0.42 ± 0.08 0.43 ± 0.22 0.09 ± 0.04 0.13 ± 0.05 0.13 ± 0.02 0.08 ± 0.03Kidney 5.85 ± 0.37 7.31 ± 0.53 3.55 ± 0.44 3.21 ± 0.45 2.18 ± 0.24 2.61± 0.51 Lungs 1.26 ± 0.24 1.12 ± 0.26 0.13 ± 0.02 0.15 ± 0.06 0.06 ± 0.000.07 ± 0.06 Blood 1.62 ± 0.34 1.59 ± 0.29 0.12 ± 0.02 0.02 ± 0.01 0.03 ±0.01 0.00 ± 0.00 Stomach 0.59 ± 0.32 0.52 ± 0.16 0.04 ± 0.01 0.07 ± 0.030.03 ± 0.01 0.04 ± 0.04 Sm. Int. 0.55 ± 0.13 2.52 ± 3.73 0.09 ± 0.010.17 ± 0.08 0.08 ± 0.01 0.04 ± 0.01 Lg. Int. 0.33 ± 0.05 0.30 ± 0.070.33 ± 0.15 0.32 ± 0.14 0.05 ± 0.01 0.07 ± 0.03

Example 4 PCR Cloning of the Mu-9 Variable Regions

Poly A mRNA was isolated from Mu-9 hybridoma cell line (≈3×10⁷ cells)using the Fast Track mRNA Isolation kit (Invitrogen, San Diego, Calif.).The first strand cDNA was reverse transcribed from poly A mRNA using thecDNA cycle kit (Invitrogen). Briefly, 1 μg of poly A mRNA was annealedto murine IgO CH1-specific primer, CH1B (5′ ACA GTC ACT GAG CTG G 3′),or murine Ck-specific primer, Ck3-BH1 (5′ GCC GGA TCC TCA CTG GAT GGTGGG AAG ATG GAT ACA 3′), at a final concentration of 1 μM at 42° for 60minutes in the presence of 1 μl of RNAse inhibitor (10 U/μl), 4.0 μl of5× reverse transcriptase buffer (500 mM Tris-HCL, pH 8.2, 200 mM KCl, 50mM MgCl₂, and 2.5 mM spermidine), 1 μl of 100 mM dNTPs, 1 μl of 80 mMsodium pyrophosphate, and 5 U of AMV reverse transcriptase. The RNA-cDNAhybrids were then denatured at 95° C. for 2 minutes. The first strandcDNAs were then used as templates to amplify the VH and Vκ sequences byPCR as described by Orlandi et al., Proc. Natl. Acad. Sci. USA 1989, 86:3833. The Vκ region was amplified using primers VK1BACK (5′-GAC ATT CAGCTG ACC CAG TCT CCA 3′) and IgKC3′ (5′-CTC ACT GGA TGG TGG GAA GAT GGATAC AGT TGG 3′). The VH region was amplified using primers VH1BACK (5′AGG T(C/G)(A/C) A(A/G)C TGC AG(C/G) AGT C(A/T)G G 3′) and CH1B. The PCRreaction mixtures containing 10 μl of the first strand cDNA product, 10μl of 10×PCR buffer (15 mM MgCl₂, 500 mM KCl, 100 mM Tris-HCl, pH 8.3,and 0.01% (w/v) gelatin), 1 μM of each primer, 16 μl of dNTPs, and 5 Uof AmpliTaq DNA polymerase (Perkin-Elmer, Applied Biosystems Division,Foster City, Calif.) were subjected to 30 cycles of PCR (denaturation at94° C. for 1 minute, annealing at 45° C. for 1 minute and polymerizationat 72° C. for 1 minute for 5 cycles, combined with denaturation at 94°C. for 1 minute, annealing at 55° C. for 1 minute, and polymerization at72° C. for 1 minute for 25 cycles). The amplified Vk and VH fragmentswere gel-purified and cloned into the TA cloning vector (Invitrogen) forsequence analyses by the dideoxytermination method. Sequences confirmedto be of immunoglobulin origin were then used to construct chimericexpression vectors using methods described by Leung et al., Hybridoma1994, 13: 469.

Nucleotide sequencing of multiple clones confirmed the isolation of oneVκ (MU9VK1) and one V_(H) (Mu9 V_(H)) sequence (FIG. 1A) The chimericMu-9 (cMu-9-1) constructed from the V_(H) and Vκ1 cloned by this RT-PCRmethod did not demonstrate any binding to CSAp antigen, suggesting thepossible existence of other “functional” V-region sequence(s) that mightbe overlooked by RT-PCR cloning procedures.

Example 5 Cloning the Mu-9 Variable Regions by cDNA Library Screening

The cDNA library was constructed from the murine Mu-9 hybridoma inpSPORT vector (Life Technologies). The first strand cDNA was synthesizedby pairing poly A mRNA from murine Mu-9 hybridoma with an oligo dTprimer-NotI adaptor (Life Technologies). After the second strandsynthesis and attachment of SalI adaptors, the cDNA pool was sizefractionated through a cDNA size fractionation column. The fractionatedcDNA was ligated to pSPORT vector and then transformed into Escherichiacoli DH5α. The library was plated onto LB-amp (100 μg/ml) plates,colonies transferred to Nytran filters (Schleicher and Schuell, Keene,N.H.), and then amplified on LB-chloramphenicol plates. The amplifiedcolonies were treated successively with 0.5 N NaOH/1.5 M NaCl for 5minutes; 1 M Tris-HCl, pH 8.0, for 5 minutes; 0.1 M Tris-HCl, pH7.5/2×SSC for 5 minutes; and finally with 2×SSC for 5-10 minutes. TheDNA was immobilized on the filters by baking at 80° C. for 30 minutes.The filters were incubated in prehybridization buffer containing 6×SSC,5×Denhardt's (0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 0.1% bovineserum albumin), 0.5% SDS, 0.05% sodium pyrophosphate, and 100 μg/mlherring sperm DNA (Life Technologies) for 2 hours at 50° C.Hybridization with the ³²P-labeled probes (MUCH-1 (5′-AGA CTG CAG GAGAGC TGG GAA GOT GTG CAC 3′) specific for murine heavy chain and MUCk-1(5′-GAA GCA CAC GAC TGA GGC ACC TCC AGA TGT 3′) specific for murinelight chain) at 10⁶ cpm/ml was done overnight at their respective Tms inthe prehybridization solution supplemented with 10% (w/v) dextransulfate (Pharmacia Biotech, Piscataway, N.J.). The filters were washedfour times in 0.2×SSC, 0.1% SDS at 37° C. for 10 minutes, twice at 42°C. for 15 minutes, and once at 50° C. for 15 minutes until theradioactivities on the filters were constant as determined with a Geigercounter. After a final rinse in 2×SSC, the wet filters were exposed toKodak XAR-5 film (Rochester, N.Y.) at 70° C. The clones that werepositive on the first screening were transferred to duplicate LB-ampplates. Duplicate Nytran filters were hybridized to the same probes asdescribed above. Only clones that positively hybridized on both thefilters were picked for further screening. For the tertiary screening,the MUCH-1-positive colonies were screened in duplicate with VHCDR3Mu9(specific for the V_(H) sequenced cloned by RT-PCR in Example 4) andMUCk-1-positive colonies were screened with VKCDR1Mu9 and VKCDR3Mu9(specific for the CDR1 and CDR3 coding sequences of Vκ-1 cloned byRT-PCR in Example 4).

All clones (25) that were confirmed to be positive with MUCH-1 were alsopositive with VHCDR3Mu9, indicating that there was only one type ofheavy chain sequence expressed in the hybridoma. Ten clones thatpositively hybridized with the VHCDR3Mu9 were sequenced and found to beidentical to the RT-PCR cloned Mu-9VH, the sequence of which isdisclosed in FIG. 1A.

Of the 34 clones that were positive for MUCk-1 in both primary andsecondary screenings only 14 hybridized to the Mu9 Vκ1-specific probes,VKCDR1Mu-9 and VKCDR3Mu9. Sequence analyses revealed that these cloneswere identical to the Mu9Vk1. Of the remaining 20 clones that werenegative for the Mu9Vk1-specific probes, 8 were subjected to DNAsequencing. Seven of these clones encoded a kappa light chain sequencewith a Vk domain that was different from Mu9Vk1 and designated asMu9Vk2, the sequence of which is disclosed in FIG. 1B. The chimeric Mu-9(cMu-9-2) constructed from the VH and Vk2 and expressed in Sp2/0 cellsshowed comparable binding affinity to CSAp antigen as that of murineMu-9 (see Example 8 and 9 for details).

Example 6 Probe Labeling for cDNA Library Screening

Oligonucleotides were synthesized on an automated 392 DNA/RNAsynthesized (Applied Biosystems) and then purified on a PD 10 column(Pharmacia Biotech). The purified oligonucleotides were labeled with[γ-³²P]ATP (Amersham, Arlington Heights, Ill.) using T4 polynucleotidekinase (New England Biolabs, Beverly, Mass.). A typical reaction mixturecontained in a final volume of 20 μl was as follows: 50 pmololigonucleotide, 60 μCi of [γ-³²P]ATP (6000 Ci/mmol), and 2 μl of 10×kinase buffer (New England Biolabs). The reaction mixture was incubatedat 37° C. for 1 hour, and the reaction was terminated with 20 μl of 0.1M EDTA. The unincorporated [γ-³²P]ATP was separated from the labeledoligonucleotide on a TE-10 Chromaspin column (Clonetech, Palo Alto,Calif.). The labeled probe was used at 10⁶ cpm/m. for hybridization.

Example 7 Transfection of SP2/0 Cells

The putative Vκ and VH sequences for Mu-9 were subcloned into the light(pKh or PKh*) and heavy chain (pG1g) expression vectors, respectively,as describe by Leung et al., supra. pKh* is essentially identical to thepKh, except that is has a XhoI/PacI linker introduced into the BstXIsite of the pKh. Because MU-9 Vκ2 obtained by cDNA screeing contained aninternal BstXI site, it was subcloned into pKh*, which could then belinearized with either NhoI or PacI for transfection.

Approximately 10 and 30 μg of linearized light (Mu-9-1pKh or Mu-9-2pKh*)and heavy (Mu-9pG1g) chain expression vectors were cotransfected intoSP2/0 cells by electroporation. Transfected cells were grown in 96-wellcell culture plates in complete medium for 2 days and then selected bythe addition of hygromycin at a final concentration of 500 U/ml.Typically, the colonies began to emerge 2-3 weeks after electroporationand were assayed for antibody secretion by enzyme-linked immunosorbentassay (ELISA). The chimeric antibodies were purified from the culturesupernatant by affinity chromatography on Protein A-Sepharose 4B column.The purified antibodies (5-μg) were analyzed by SDS-PAGE on a 4-20%gradient gel under reducing conditions.

Example 8 Mu-9 Direct Binding Assay

ELISA microtiter plates were coated with a void volume fraction of GW-39tumor extracts (which contains the CSAp antigen) eluted from a Sepharose4B-CL column and left overnight at 4° C. The next day, the nonspecificbinding was blocked with phosphate-buffered saline (PBS) containing 1%BSA and 0.05% Tween 20. Chimeric antibody supernatant (100 μl) orpurified antibody (0-1 μg/ml) was added and incubated at roomtemperature for 1 hour. Unbound proteins were removed by washing sixtimes with wash buffer (PBS containing 0.05% Tween 20). Purified murineMu-9 protein was used as the standard. The bound antibodies were allowedto react with peroxidase-conjugated goat anti-human IgG, Fcfragment-specific (Jackson ImmunoResearch, West Grove Pa.) andperoxidase conjugated goat anti-mouse IgG, Fc fragment-specificantibodies (Jackson ImmunoResearch). After washing the plate six timeswith wash buffer, 100 μl of OPD substrate solution (10 mg oforthophenylenediamine dihydrochloride (Sigma, St. Louis, Mo.) in 25 mlof 0.32×PBS and 0.12% H₂O₂) was added to each well. The color wasdeveloped in the dark for 1 hour, the reaction was stopped with 50 μl of4NH₂SO₄, and the absorbance at 490 nm was measured in a Dynatech platereader (Dynatech Labs, Sussex, UK).

Direct binding of a cMu-9 (cMu-9-2) to CSAp antigen occurred, as shownin FIG. 5( a). The binding profile of cMu-9-2 was virtuallysuperimposable on that of the murine Mu-9. These data demonstrated thatthe immunoreactivity of cMu-9-2 is comparable to that of murine Mu-9.The DNA and amino acid sequences of the functional cMu-9 Vκ and V_(H)are shown in FIGS. 2A and 2B, respectively.

Example 9 Competitive Binding Assay

Murine Mu-9 IgG was conjugated with horseradish peroxidase (HRP)(Sigma). The peroxidase-conjugated Mu-9 was first tested for binding onmicrowells coated with CSAp antigen, and the optimum concentration wasdetermined to be 0.2 μg/ml. Peroxidase-conjugated Mu-9 was mixed withvarious concentrations of either murine or chimeric Mu-9 (0-50 μg/ml)before addition to the antigen-coated wells. Binding of theperoxidase-conjugated Mu-9 to the antigen in the presence of thecompeting antibodies was measured at 490 nm after the addition of thesubstrate as described earlier.

FIG. 5( b) displays the results of the competitive binding assay. MurineMu-9 and cMu-9-2 competed equally well with the binding ofHRP-conjugated Mu-9 to the CSAp antigen. These data demonstrated thatthe immunoreactivity of cMu-9-2 is comparable to that of murine Mu-9.

Example 10 Choice of Human Frameworks and Sequence Design for theHumanization of Mu-9 Monoclonal Antibody

By comparing the variable (V) region framework (FR) sequences of Mu9 tothat of human antibodies in the Kabat data base, the FRs of Mu9VH and Vκwere found to exhibit the highest degree of sequence homology to that ofthe human antibodies, EU V_(H) and WOL Vκ, respectively. In FIGS. 3A and3B, the amino acid sequences of the Mu9VH and Vκ Abs are aligned andcompared with the corresponding human sequences. Therefore, the FR^(s)of EU VH and WOL Vκ were selected as the human frameworks onto which theCDRs for Mu-9V_(H) and Vκ were grafted, respectively. The FR4 sequenceof NEWM, however, rather than that of EU, was used for the humanizationof Mu9 heavy chain (FIG. 3A). A few amino acid residues in Mu9 FR^(s)that are close to the putative CDRs were maintained in hMu9 based on theguideline described previously (Qu et al., Clin. Cancer Rec.5:3095s-3100s (1999)). These residues are L37, V58 and Q100 of Vκ (FIG.3B) and Y27, T30, K38, R40, I48, K66, A67, K74, T93, R94 and G103 of VH(FIG. 3A). The gene sequences of hMu9VH and Vκ were then designed andshown with the amino acid sequences in FIGS. 4A and 4B, respectively.

Example 11 PCR/gene Synthesis of the Humanized V Genes

The strategy as described by Leung et al. (Leung et al., 1994)) was usedto construct the designed Vκ and VH genes for hMu-9 using a combinationof long oligonucleotide systheses and PCR. Each variable chain wasconstructed in two parts, a 5′- and 3′-half, designated as “A” and “B,”respectively. Each half was produced by PCR amplification of a singlestrand synthetic oligonucleotide template with two short flankingprimers, using Taq polymerase. The amplified fragments were first clonedinto the pCR4TA cloning vector from Invitrogen (Carlsbad, Calif.) andsubjected to DNA sequencing. The templates and primer pairs are listedas follows:

Template Primers PCR product Oligo G Oligo 14/Oligo 15 hMu9VHA Oligo HOligo 16/Oligo 17 hMu9VHB Oligo J Oligo 18/Oligo 19 hMu9VkA Oligo KOligo 20/Oligo 21 hMu9VkB

Heavy Chain

For constructing the full-length DNA of the hMu9VH sequence, Oligo G(102 mer) and H (179 mer) were synthesized on an automated RNA/DNAsynthesizer (Applied Biosystems). Oligo G sequence represents the minusstrand of hMu9VH domain complementary to nt 19 to 120:

5′-AGGTCTCTGT TTTACCCAGG TAATAACATA CTCAGTGAAG GTGTATCCAG AAGCCTTGCAGGAGACCTTC ACTGAGCTCC CAGGCTTTTT CACCTCAGCT CC-3′

Oligo H sequence represents the nt 147 to 325 of hMu9VH domain:

5′-GATTTATCCT GGAAGTGGTA GTACTTCCTA CAATGAAAAG TTCAAGGGCA AGGCCACAATCACTGCTGAC AAATCCACTA ACACAGCCTA CATGGAGCTC AGCAGCCTGA GATCTGAGGACACTGCGTTC TATTTCTGTA CAAGAGAGGA TCTTGGGGGC CAAGGGTCTC TGGTCACCG-3′

Oligo G and H were cleaved from the support and deprotected by treatmentwith concentrated ammonium hydroxide. After samples were vacuum-triedand resuspended in 100 μl of water, incomplete oligomers (less than100-mer) were removed by centrifugation through a ChormaSpin-00 column(Clontech, Palo Alto, Calif.). All flanking primers were preparedsimilarly, except ChromaSpin-30 columns were used to remove synthesisby-products. 1 μl of ChromaSpin column purified Oligo G was PCRamplified in a reaction volume of 100 μl containing 10 μl of 10×PCRbuffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, and 0.01%(w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), 250 μM of eachdNTP, 200 nM of Oligo14 (5′-GTGCAGCTGC AGCAGTCAGG AGCTGAGGTG-3′) andOligo 15 (5′-ACTCTAGACC CTGTCCAGGT CTCTGTTTTA CCCAGGTAAT AACATA-3′), and5 units of Taq DNA polymerase (Perkin Elmer Cetus). This reactionmixture was subjected to 30 cycles of PCR reaction consisting ofdenaturation at 94° C. for 1 min, annealing at 50° C. for 1.5 min, andpolymerization at 72° C. for 1.5 min. Oligo H was PCR-amplified by theprimer pair Oligo 16 (5′-GGTCTAGAGT GGATTGGAGA GATTTATCCT GGAAGTGGTAGTACTT-3′) and Oligo 17 (5′-TGAAGAGACG GTGACCAGAG ACCCTTGGCC CCCAAGATCCTCTCTTGTAC AGAAATAGAA CGC-3′) under similar condition. Resulting PCRfragments, VHA and VHB were purified on 2% agarose (BioRad, Richmond,Calif.). Unique restriction sites were designed at the ends of eachfragment to facilitate joining through DNA ligation. The amplified VHAfragment contained a PstI restriction site, CTGCAG, at its 5′-end and aXbaI restriction site, TCTAGA, at the 3′-end. The amplified VHB fragmentcontained a XbaI restriction site at its 5′-end and a BstEII restrictionsite, GGTCACC, at the 3′-end. Assembly of the full-ength V_(H) chain wasaccomplished by restriction enzyme digestion of each fragment with theappropriate 5′- and 3′-enzymes and ligation into the VHpBS vector (Leunget al., Hybridoma, 13:469 (1994)) previously digested with PstI andBstEII. The resulting ligated product contains the A fragment ligated tothe PstI site, the B fragment to the BstEI site, and the A and Bfragments joined together at the XbaI site (FIG. 4A). Upon confirmationof a correct open reading frame by DNA sequencing, the intact VH genesequence along with the promoter and the secretion signal peptide codingsequence was removed from VHpBS as a HindIII to BamHI fragment andligated into the VHpG1g expression vector (Leung et al., Hybridoma,13:469 (1994)), resulting in hMu9VHpG1g.

Light Chain

For the construction of VK, the long oligonucleotide templatessynthesized were Oligo J (130 mer) representing the minus strand ofhMu9Vκ domain complementary to nt 21 to 150:

5′-CCTTGGAGCC TGGCCTGGTT TCTGCAGGTA CCATTCTAAA TAGGTGTTGC CATTACTATGCACAATGCTC TGACTAGACC TGCAAGACAG AGTGGCTCGC TCTCCAGGAC TGAGGGACAGGGTGCCTGGG-3′

and Oligo K (150 mer) representing the nt 151 to 300 of hMu9Vκ domain:

5′-CTCCTGATCT ACAAAGTTTC CAACCGATTT TCCGGAGTCC CAGACAGGTT CAGTGGCTCTGGATCAGGGA CAGATTTCAC ACTTACTATC AGCAGACTGG AGCCTGAGGA TTTTGCTGTGTATTACTGCT TTCAAGGTTC ACGTGTTCCG-3′

These oligos were PCR-amplified by their respective primer pairs aslisted:

Oligo 18 5′-GATATCCAGC TGACCCAATC CCCAGGCACC CTGTCCCTCA GTCCTGGAG-3′Oligo 19 5′-AGATCAGGAG CCTTGGAGCC TGGCCTGGTT TCTGCA-3′ Oligo 205′-TACCTGCAGA AACCAGGCCA GGCTCCAAGG CTCCTGATCT ACAAAGTTTC CAACCG-3′Oligo 21 5′-TTAATCTCCA CCTTGGTCCC CCCTCCGAAC GTGTACGGAA CACGTGAACCTTGAAAGCAG TAATACA-3′

The same construction method as done for VH was carried out for Vκ, withthe following modifications: the 5′-end restriction site of the Afragments was PvuII (CAGCTG) and the 3′-end restriction site of Bfragments was BglII (AGATCT). These fragments were joined together uponligation into the VKpBR vector at a common PstI site (CTGCAG), resultingin full-length Vκ sequence (FIG. 4B) and confirmed by DNA sequencing.The assembled Vκ gene was subcloned as HindIII-BamHI restrictionfragment into the light expression vector, resulting in hMu9VKpKh.

Example 12 Transfection, Expression and Binding Activity Assays for hMu9

The methods for expression and binding activity assays for hMu9 weresame as described for cMu9. Approximately 10 and 30 μg of linearizedhMu9VkpKh and hMu9VHpG1g were co-transfected into SP2/0 cells byelectroporation. Transfected cells were grown in 96-well cell cultureplates in complete medium for 2 days and then selected by the additionof hygromycin at a final concentration of 500 U/ml. Typically, thecolonies began to emerge 2-3 weeks after electroporation and wereassayed for antibody secretion by enzyme-linked immunosorbent assay(ELISA). The chimeric antibodies were purified from the culturesupernatant by affinity chromatography on Protein A-Sepharose 4B column.The purified antibodies (5 μg) were analyzed by SDS-PAGE on a 4-20%gradient gel under reducing conditions.

Direct binding assay showed that the purified hMu-9 bound to CSApantigen. The binding affinity of hMu-9 was compared in a competitivebinding assay as described in Example 6. FIG. 6 displays the results ofthe competitive binding assay. hMu-9 or murine Mu-9 competed equallywell with the binding of HRP-conjugated Mu-9 to the CSAp antigen. Thesedata demonstrated that the immunoreactivity of hMu-9 is comparable tothat of murine Mu-9.

Example 13 Therapy of a Patient with ⁹⁰Y-labeled Humanized Mu-9 Antibody

A 62-year-old man, with a history of Dukes° C. rectal carcinoma that wasresected 3 years earlier, at which time radiation therapy followed by5-fluorouracil/folinic acid chemotherapy were given, began showing arise in his plasma CEA titer over the last 6 months, reaching a level of30 ng/mL. The patient, who was seeing his oncologist twice annually,learned of this result and underwent various diagnostic proceduresbecause of a suspected recurrence. It was found, by computedtomorgraphy, that there were two metastases present in his liver, onebeing 3 cm in diameter in his right lobe, and the other being somewhatsmaller in the left lobe, close to the interlobe ligament. The patientopted not to undergo chemotherapy, and was then given a dose of 25 mCi⁹⁰Y conjugated to the humanized Mu-9 antibody, given at a protein doseof 50 mg by intravenous infusion over a period of 2 hours. This therapywas then repeated two months later. The patients had a drop of his whiteblood cells and platelets, measured 24 weeks after the last therapyinfusion, but recuperated at the 8-week post-therapy evaluation. Thecomputed tomography findings at 3 months post-therapy revealed 40%shrinkage of the major tumor metastasis of the right liver lobe, and alesser reduction in the left-lobe tumor. At this time, the patient'sblood CEA dropped to 15 ng/mL. At the 6-month follow-up, his tumorlesions had been reduced, in two-diameter CT-measurements, by about 70percent, his plasma CEA was at 8 ng/mL, and his general condition wasfine, with no apparent toxicity or adverse events related to thetherapy. The patient is now 9 months post-therapy with no change in thesize of his liver metastases and a stable serum CEA titer at about 5-8ng/mL. He is being followed every 3 months, so that if the diseasebegins to grow, he is scheduled to receive another course of thisradioimmunotherapy, followed by a course of naked Mu-9 antibody, at aweekly dose of 300 mg/r², once weekly for 6 weeks, concomitantly with atherapy course of irinotecan (CPT-11).

VII. REFERENCES

All references cited, as well as references cited by the referencescited herein, are hereby incorporated herein by reference in theirentireties.

Additional references of interest, as well as references cited therein,include the following, and are hereby incorporated herein by referencein their entireties:

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1. A method of delivering a diagnostic and/or therapeutic agent to aCSAp-expressing tumor comprising: a) obtaining an anti-CSAp antibody orfragment thereof, wherein the anti-CSAp antibody binds to the sameepitope of CSAp as an Mu-9 antibody, wherein the Mu-9 antibody comprisesthe light chain complementarity determining region (CDR) sequences CDR1(RSSQSIVHSNGNTYLE, SEQ ID NO: 1), CDR2 (KVSNRFS, SEQ ID NO:2) and CDR3(FQGSRVPYT, SEQ ID NO:3) and the heavy chain CDR sequences CDR1 (EYVIT,SEQ ID NO:4), CDR2 (EIYPGSGSTSYNEKFK, SEQ ID NO:5) and CDR3 (EDL); b)conjugating said anti-CSAp antibody or fragment to at least onediagnostic and/or therapeutic agent; and e) administering the conjugatedanti-CSAp antibody or fragment thereof to a subject in need thereofwherein said administration is effective to deliver said at least onediagnostic or therapeutic agent to said CSAp-expressing tumor.
 2. Themethod of claim 1, wherein the anti-CSAp antibody is a humanizedanti-CSAp antibody.
 3. The method of claim 2, wherein the humanizedanti-CSAp antibody comprises the amino acid sequences of h-Mu-9VH (SEQID NO:38) and hMu-9VK (SEQ ID NO:40).
 4. A method of diagnosing ordetecting a CSAp-expressing cancer comprising: a) obtaining an anti-CSApantibody or fragment thereof, wherein the anti-CSAp antibody binds tothe same epitope of CSAp as an Mu-9 antibody, wherein the Mu-9 antibodycomprises the light chain CDR sequences CDR1 (RSSQSIVHSNGNTYLE, SEQ IDNO:1), CDR2 (KVSNRFS, SEQ ID NO:2) and CDR3 (EQGSRVPYT, SEQ ID NO:3) andthe heavy chain CDR sequences CDR1 (BYVIT, SEQ ID NO:4), CDR2(EIYPGSGSTSYNEKFK. SEQ ID NO:5) and CDR3 (EDL); b) conjugating saidanti-CSAp antibody or fragment to at least one diagnostic agent; c)administering the conjugated anti-CSAp antibody or fragment thereof to asubject in need thereof; and d) detecting conjugated anti-CSAp antibodyor fragment bound to CSAp, wherein the presence of bound antibody orfragment is diagnostic for or indicates the presence of aCSAp-expressing cancer.
 5. A method of treating a CSAp-expressing cancercomprising: a) obtaining an anti-CSAp antibody or fragment thereof,wherein the anti-CSAp antibody binds to the same epitope of CSAp as anMu-9 antibody, wherein the Mu-9 antibody comprises the light chain CDRsequences CDR1 (RSSQSTVHSNGNTYLE, SEQ ID NO:1), CDR2 (KYSNICS, SEQ IDNO:2) and CDR3 (FQGSRVPYT, SEQ ID NO:3) and the heavy chain CDRsequences CDR1 (BYVIT, SEQ ID NO:4), CDR2 (EIYPGSCSTSYNEKEK, SEQ IDNO:5) and CDR3 (EDL); and b) administering the anti-CSAp antibody orfragment thereof to a subject with a CSAp-expressing cancer.
 6. Themethod of claim 5, wherein said anti-CSAp antibody or fragment is anaked antibody or fragment.
 7. The method of claim 6, further comprisingadministering at least one therapeutic agent to said subject.
 8. Themethod of claim 7, wherein said therapeutic agent is selected from thegroup consisting of a an immunomodulator, a cytokine a hormone, ahormone antagonist, an enzyme, an enzyme inhibitor, a photoactivetherapeutic agent, a cytotoxic drug, an angiogenesis inhibitor, anantibody that does not bind CSAp and a combination thereof.
 9. Themethod of claim 5, further comprising conjugating at least onetherapeutic agent to the anti-C SAp antibody or fragment thereof priorto said administering step.
 10. The method of claim 9, whereinadministering said conjugated antibody or fragment thereof to saidsubject is effective to treat said cancer.
 11. The method of claim 10,wherein said cancer is selected from the group consisting ofgastrointestinal cancer, ovarian cancer, colorectal cancer andpancreatic cancer.
 12. The method of claim 9, wherein said therapeuticagent is selected from the group consisting of a radionuclide, boron,gadolinium, uranium, an immunomodulator, a cytokine a hormone, a hormoneantagonist, an enzyme, an enzyme inhibitor, a photoactive therapeuticagent, a cytotoxic drug, a toxin, an angiogenesis inhibitor, a secondantibody or fragment thereof and a combination thereof.
 13. The methodof claim 12, wherein said therapeutic agent is a drug or toxin.
 14. Themethod of claim 13, wherein said drug is selected from the groupconsisting of antimitotic, alkylating, antimetabolite,angiogenesis-inhibiting, apoptotic, alkaloid, COX-2-inhibiting andantibiotic agents and combinations thereof.
 15. The method of claim 13,wherein said drug is selected from the group consisting of nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas,triazenes, folic acid analogs, anthracyclines, taxanes, COX-2inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes,epipodophyllotoxins, platinum coordination complexes, vinca alkaloids,substituted ureas, methyl hydrazine derivatives, adrenocorticalsuppressants, hormone antagonists, enzyme inhibitors, endostatin,taxols, camptothecins, doxorubicins and their analogs, and a combinationthereof.
 16. The method of claim 13, wherein said toxin is selected fromthe group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease(RNase), DNase 1, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.
 17. The method of claim 12, wherein saidradionuclide is selected from the group consisting of Ac-225, Ag-111,As-77, At-211, At-217, Au-198, Au-199, Bi-213, Bi-212, Bi-211, Ur-80m,Co-58, Cu-64, Cu-67, Dy-152, Er-169, Fe-59, Fm-255, Fr-221, Ga-67,Ho-166, Ho-161, I-125, I-131, In-111, Ir-194, Ir-192, Lu-177, Mo-99,Os-189m, P42, P-33, Pb-211, Pb-212, Pd-109, Pm-149, Po-215, Pr-142,Pr-143, Pt-109, Ra-223, Re-186, Re-188, Re-189, Rh-10S, Rh-103m, Rn-219,Sb-19, Sc-47, Se-75, Sm-153, Sr-89, Tb-161, Tc-99m, Y-90, andcombinations thereof.
 18. The method of claim 12, wherein said secondantibody or fragment thereof binds to a tumor-associated antigen. 19.The method of claim 12, wherein said therapeutic agent is an enzyme andsaid method further comprises administering a prodrug to said subject.20. The method of claim 19, wherein said prodrug is selected from thegroup consisting of epirubicin glucuronide, CPT-11, etoposideglucuronide, daunomicin glucuronide and doxorubicin glucuronide.